The Seneca tribe of Native Americans lived in and around Rochester until losing claim to most of this land in the Treaty of Big Tree in 1797. Settlement before the Seneca tribe is unknown.

Rochester's development followed the American Revolution, and forced cession of their territory by the Iroquois after Britain's defeat. Allied with the British, four major Iroquois tribes were forced out of New York. As a reward for their loyalty to the British crown, they were given a large land grant on the Grand River in Canada.

Rochester was founded shortly after the American Revolution by a wave of English-Puritan-descended immigrants from New England, who were looking for new agricultural land. They were the dominant cultural group in Rochester for over a century. On November 8, 1803, Colonel Nathaniel Rochester (1752–1831), Major Charles Carroll, and Colonel William Fitzhugh, Jr. (1761–1839), all of Hagerstown, Maryland, purchased a 100-acre (40-ha) tract from the state in western New York along the Genesee River. They chose the site because its three cataracts on the Genesee offered great potential for water power. Beginning in 1811, and with a population of 15, the three founders surveyed the land and laid out streets and tracts. In 1817, the Brown brothers and other landowners joined their lands with the Hundred Acre Tract to form the village of Rochesterville.

By 1821, Rochesterville was the seat of Monroe County. In 1823, it consisted of 1,012 acres (4 km²) and 2,500 residents, and the Village of Rochesterville became known as Rochester. Also in 1823, the Erie Canal aqueduct over the Genesee River was completed, and the Erie Canal east to the Hudson River was opened. In the early 20th century, after the advent of railroads, the presence of the canal in the center city was an obstacle; it was rerouted south of Rochester by 1918 when the Barge Canal was completed. By 1830, Rochester's population was 9,200, and in 1834, it was rechartered as a city.

Rochester was first known as "the Young Lion of the West", and then as the "Flour City". By 1838, it was the largest flour-producing city in the United States. Having doubled its population in only 10 years, Rochester became America's first "boom town".

In 1830–31, Rochester experienced one of the nation's biggest Protestant revivalist movements, led by Charles Grandison Finney. The revival inspired other revivals of the Second Great Awakening. A leading pastor in New York, who was converted in the Rochester meetings, gave this account of Finney's meetings there: "The whole community was stirred. Religion was the topic of conversation in the house, in the shop, in the office, and on the street. The only theater in the city was converted into a livery stable; the only circus into a soap and candle factory. Grog shops were closed; the Sabbath was honored; the sanctuaries were thronged with happy worshippers; a new impulse was given to every philanthropic enterprise; the fountains of benevolence were opened, and men lived to good."

By the mid-19th century, as the center of the wheat-processing industry moved west with population and agriculture, the city became home to an expanding nursery business, giving rise to the city's second nickname, the Flower City. Nurseries ringed the city, the most famous of which was started in 1840 by immigrants Georg Ellwanger from Germany and Patrick Barry from Ireland.

In 1847, Frederick Douglass founded the abolitionist newspaper, the North Star in Rochester. A former slave and an antislavery speaker and writer, he gained a circulation over 4,000 readers in the United States, Europe, and the Caribbean. The North Star served as a forum for abolitionist views. The Douglass home burnt down in 1872, but a marker for it is in Highland Park off South Avenue.

Susan B. Anthony, a national leader of the women's suffrage movement, was from Rochester. The Nineteenth Amendment to the United States Constitution, in 1920, which guaranteed the right of women to vote, was known as the Susan B. Anthony Amendment because of her work toward its passage, which she did not live to see. Anthony's home is a National Historic Landmark known as the National Susan B. Anthony Museum and House.

At the end of the 19th century, anarchist Emma Goldman lived and worked in Rochester for several years, championing the cause of labor in Rochester sweatshops. Rochester also had significant unrest in labor, race, and antiwar protests.

After the Civil War, Rochester had an expansion of new industries in the late 19th century, founded by migrants to the city, including inventor and entrepreneur George Eastman, who founded Eastman Kodak, and German immigrants John Jacob Bausch and Henry Lomb, who launched Bausch & Lomb in 1861. Not only did they create new industries, but Eastman also became a major philanthropist, developing and endowing the University of Rochester, its Eastman School of Music, and other local institutions.

In the early 20th century, Rochester became a center of the garment industry, particularly men's fashions. It was the base of Bond Clothing Stores, Fashion Park Clothes, Hickey Freeman, and Stein-Bloch and Co. Carriagemaker James Cunningham and Sons founded the pioneer automobile company Cunningham.

Rochester's black population tripled to more than 25,000 during the 1950s. Casually employed by the city's iconic industries, most African Americans in the city held low-pay and low-skill jobs, and lived in substandard housing. Discontent exploded in the 1964 Rochester race riot. Triggered by the attempted arrest of a 19-year-old intoxicated black male at a street block party, order was restored after three days, and only after Governor Nelson Rockefeller called out the New York National Guard. By the time the disturbance was over, four were dead (three in a helicopter crash) and 350 were injured. Almost a thousand people were arrested and 204 stores were either looted or damaged.

In the wake of the riots, the Rochester Area Churches, together with black civil rights leaders, invited Saul Alinsky of the Industrial Areas Foundation to help the community organize. With the Reverend Franklin Florence, who had been close to Malcolm X, they established FIGHT (Freedom, Integration, God, Honor, Today), which successfully brought pressure to bear on Eastman Kodak to help open up employment and city governance.

The population reached 62,386 in 1870, 162,608 in 1900, and 295,750 in 1920. By 1950, the population had reached a high of 332,488. In 1950, the Census Bureau reported Rochester's population as 97.6% White and 2.3% Black. With industrial restructuring in the later 20th century, and the decline of industry and jobs in the area, by 2018, the city's population had declined to 206,284 (although the metropolitan area was considerably larger) with 46.58% recorded as White and 40.71% as Black or African American.

According to the 2010 census, the city's population was 43.7% White or White American, 41.7% Black, 0.5% American Indian and Alaska Native, 3.1% Asian, 0.0% Native Hawaiian and Other Pacific Islander, 6.6% from some other race and 4.4% from two or more races. 16.4% of the total population were Hispanic or Latino of any race, mostly made up of Puerto Ricans.Non-Hispanic Whites were 37.6% of the population in 2010, compared to 80.2% in 1970.

Over the course of the past 50 years, Rochester has become a major center for immigration, particularly for arrivals from Eastern and Southeastern Europe, sub-Saharan Africa, and the Caribbean. Rochester had the highest percentage of Puerto Ricans of any major city in the United States in 2013, one of the four largest Turkish American communities, one of the largest Jamaican American communities in any major U.S city and a large concentration of Polish Americans along with nearby Buffalo, New York. Rochester's Bhutanese and Nepalese communities are among the largest (top 3) in the United States, concentrated primarily in Jones Square and Edgerton with growth fueled by recently arrived migrants and refugees. In addition, Rochester was ranked number 9 in the nation for the largest Italian population in the United States in 2018.

In 1997, Rochester was reported to have the largest per capita deaf population in the United States, likely because it is home to the National Technical Institute for the Deaf.

In 2010, of 88,999 households, 30.0% had children under 18 living with them, 25.1% were married couples living together, 23.3% had a female householder with no husband present, and 47.0% were not families. Of all households, 37.1% were made up of individuals, and 9.2% had someone living alone 65 or older. The average household size was 2.36, and the average family size was 3.19. The age distribution was 28.1% under 18, 11.6% from 18 to 24, 32.2% from 25 to 44, 18.1% from 45 to 64, and 10.0% who were 65 or older. The median age was 31. For every 100 females, there were 91.6 males. For every 100 females 18 and over, there were 87.3 males.

The median income for a city household was $27,123, and for a family was $31,257. Males had a median income of $30,521, versus $25,139 for females. The per capita income for the city was $15,588. About 23.4% of families and 25.9% of the population were below the poverty line, including 37.5% of those under age 18 and 15.4% of those age 65 or over.

By the 1920s and 1930s, Rochester's population was roughly half Protestant and half Catholic, although a significant Jewish population also was present. In 1938, the city had 214 religious congregations, two-thirds of which had been founded after 1880. At that time, the city added, on average, 2.6 new congregations per year, many founded by immigrants from Southern and Eastern Europe. During peak immigration from 1900 to 1920, dozens of churches were established, including four Roman Catholic churches with Italian clergy, three Roman Catholic churches with Slavic clergy, a Polish Baptist church, 15 Jewish synagogues, and four small Italian Protestant mission churches (Baptist, Evangelical, Methodist, and Presbyterian). Additionally, several Buddhist temples are in the city, one Cambodian, two Lao, and one Vietnamese.

In 2012, Rochester had 2,061 reported violent crimes, compared to a national average rate of 553.5 violent crimes in cities with populations larger than 100,000. That same year, Rochester had 827 personal-crime incidents and 11,054 property-crime incidents.

In 2018, Rochester reported 28 murders (13.9 per 100,000 residents). In 2012, 95 sexual assaults, 816 robberies, 1,104 aggravated assaults, 2,978 burglaries, 7,694 larceny thefts, 111 forcible rape, 622 auto thefts, and 152 acts of arson occurred.

Notable individuals who were born in and/or lived in Rochester include former NBA Basketball Player John Wallace, American social reformer and women's rights activist Susan B. Anthony, African-American social reformer and abolitionist Frederick Douglass, Kodak founder George Eastman, actors Philip Seymour Hoffman, Kristen Wiig, and John Lithgow, Paychex founder Tom Golisano, Olympic soccer player Abby Wambach, opera singer Renée Fleming, jazz singer Cab Calloway, jazz musician Chuck Mangione, composer Howard Hanson, mandopop singer Wang Leehom, actress Olympic swimmer Ryan Lochte, NHL ice hockey player Ryan Callahan, YouTube personalities Jenna Marbles and Andrew Rea (Binging with Babish), RuPaul's Drag Race star Pandora Boxx, UFC Light Heavyweight Champion Jon Jones, and former NFL draft picks Kevin McMahan and Chandler Jones.^{[citation needed]}