Bartram was born in Darby Township into a Quaker family. He travelled extensively in the eastern United States collecting plants, from Lake Ontario in the north, to Florida in the south and the Ohio River in the west. Many of his acquisitions were transported to collectors in Europe.
He founded Bartram Botanical gardens in Philadelphia. He was one of the co-founders, with Benjamin Franklin, of the American Philosophical Society in 1742.
In 1765 George III made Bartram Royal Botanist, a post he held until his death.