While playing as Desmond, players can find a set of bright red footprints in Eagle Vision while in Monterigionnni (leading from the Villa to the sewers). A popular opinion by gamers was that this was a clue to indicate that either Lucy, Shaun or Rebecca may be a Templar. In truth, the footprints were accidentally left in by the programmers when beta testers got lost during the initial missions at the Villa.
It has been confirmed by the programmers and writers of the game that while Altaïr (of the first game in the franchise) and Ezio are both ancestors of Desmond Miles, they are both from different bloodlines and not related to each other.
The characters of Lucy Stillman and Shaun Hastings are modeled to resemble the actors who voice them (Kristen Bell and Danny Wallace).
Almost all of the equipment in the Sanctuary has the Abstergo (Templar) Logo on it.
This was the first Assassin's Creed video game that gave the ability to recruit assassins.