The American Civil War (1861 – 1865) was an armed military conflict in the United States of America. The war began when 11 Southern states elected to secede from the rest of the country in 1861 to form the Confederate States of America for multiple reasons. The war ended four years later with the victory of the Northern states.
Several causes, such as the issues of states' rights and slavery, led to the conflict, which eventually culminated with the assassination of President Abraham Lincoln after the war's end by the Templar-affiliated John Wilkes Booth, who in turn was tracked down and killed by Assassins.
The American Civil War was a point of interest for the Animus Project, and a number of Subjects chosen for it, including Clay Kaczmarek and Desmond Miles, had ancestors who had participated in the conflict. Additionally, Abstergo Entertainment supervisor Melanie Lemay also had a great-great-grandfather who was known to have come into contact with Lincoln a number of times as a Union soldier during the conflict. Not to mention that the Northern and Southern branches of the American Brotherhood fought on opposite sides of the conflict as much as they did with the Templars. 
The Assassin Varius, lived in New York City during the civil war. In 1863, the mentor tasked Varius to steal a Dagger of Eden and to hand it to General Ulysses S. Grant, the dagger managed to reach General Grant. After retrieving the Dagger, Varius trained a young Assassin named Eliza to deliver it to Grant.
- In 2016, Callum Lynch was noted to have Assassin ancestors who fought on the Confederate side during the war.
- Assassin's Creed II (mentioned only)
- Assassin's Creed: Revelations (mentioned only)
- Assassin's Creed IV: Black Flag (mentioned only)
- Assassin's Creed: Last Descendants (first appearance)
- Assassin's Creed: The Movie (mentioned only)