Following the conclusion of the "Ezio Trilogy" with Assassin's Creed Revelations, Assassin's Creed III shifts focus towards a new historical time period: 18th-century Colonial America. The game follows two new protagonists: Haytham Kenway, a British nobleman and high-ranking member of the Templar Order, who is sent to America to establish a strong Templar presence on the continent and find an ancient temple built by the First Civilization; and Ratonhnhaké:ton, also known as Connor, Haytham's half-Mohawk son, who joins the Assassins to avenge the burning of his village and his mother's death and stop the Templars from gaining control of the colonies.

Haytham's story serves as a prologue to the main narrative, and takes place from 1754 to 1755, at the onset of the French and Indian War, which paved the way for the American Revolution. Connor's story spans two decades of his life, from his childhood in 1760 to 1783, and involves major events from the American Revolution and the Revolutionary War seen from Connor's perspective.[16] Boston and New York City are cities that can be explored, as well as the American Colonial Frontier, spanning forest, cliffs, rivers, Connor's village, and the settlements of Lexington and Concord.

The player can hunt small and large animals, and approximately one-third of the story takes place in the Frontier. The city of Philadelphia can also be visited at one point during the game, as can The Caribbean during several naval missions. The Eastern seaboard is explorable via Connor's captaining his naval warship, the Aquila. Like the previous games in the series, the story includes segments set during the modern day, in 2012, which again follow Desmond Miles and his efforts to save humanity from an impending disaster.

These segments have been expanded significantly, and involve actual missions that the player must complete to advance the narrative. Locations visited during the modern-day segments include a skyscraper in Manhattan, New York; a stadium in São Paulo, Brazil; and Abstergo's facility in Rome, Italy.