During the early 16th century, it was the base of the Knights Hospitaller, a chivalric order closely aligned with the Templars. As a result, the island also doubled as a Templar stronghold, and at least one Templar, the deacon Cyril, hailed from the island. The city was a focal point in the Assassin-Templar War and one of many cities in the Mediterranean Sea that the two factions vied control over.
In 1511, Hospitallers captured a number of Assassins and imprisoned them in their Palace of the Grand Master, prompting a rescue mission by the Assassin Mentor Ezio Auditore da Firenze who sent a team of Ottoman Assassins from Constantinople. While the Assassins were successful in retrieving their comrades, the Hospitallers reacted by slaying the Master Assassin Castor. A full-fledged Assassin assault followed that saw the destruction of the Hospitaller stronghold and the establishment of an Assassin Guild on the island.
In spite of this, the Rhodes continued to be the sovereign territory of the Knights Hospitaller until 1522. That year, a massive invasion by the Sultan Suleiman I of the Ottoman Empire, an Assassin ally, definitively ousted the Hospitallers from the island, sparking a centuries-long feud between the Ottomans and Hospitallers.
- Rhodes is initially unavailable for Mediterranean Defense in Assassin's Creed: Revelations, but can be unlocked by playing a match on each Animi training map set in the city; these maps are Knight's Hospital, Souk and Ippokratous.
- Because of this, Rhodes is exempt from the the achievement Armchair General that is granted to the player once the Assassins have control over all of the cities.
- Rhodes remains entirely unavailable in Mediterranean Defense in Assassin's Creed: The Ezio Collection due to the absence of multiplayer modes.