Leptospiral agglutinins have been measured in several species of chelonians in studies dating back to the1960s. However, despite the fact that sea turtles are one of the most charismatic and endangered groups of chelonians, only a single study has measured leptospiral antibodies in this group. Serum samples collected from hawksbill sea turtles (Eretmochelys imbricata) (n = 10) and green sea turtles (Chelonia mydas) (n = 10) from two locations from the Gulf of Mexico were analyzed to detect Leptospira interrogans using the microagglutination test. Hawksbill and green sea turtles tested positive to five and seven serovars, respectively, with the serovars Grippotyphosa, Bratislava, and Canicola presenting a prevalence ≥50% in both species. Titers ranged from 1:50 to 1:400. These and previous results demonstrate that terrestrial, freshwater, and sea turtles are exposed to Leptospira at some stage of their life. The role of reptiles in the epidemiology, persistence, and transmission of leptospirosis is still controversial; however, there is evidence that some species of turtles can disseminate them in the environment. The detection of Leptospira is of sanitary importance as it is a cosmopolitan zoonotic disease. Preventive measures should be taken by people working in sea turtle camps and aquariums to control and reduce their risk of exposure.