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Habitat loss and degradation in the Mexican Caribbean, caused by the development of tourism, have decreased the potential nesting area for the American crocodile (Crocodylus acutus) and have fragmented the populations of the Yucatan peninsula. Our study investigated Wve populations (three continental: North, South, Sian Ka'an, and two insular: Cozumel, Banco Chinchorro) of C. acutus in the Mexican Caribbean using seven diVerent inter simple sequence repeat (ISSR) markers as tools for genetic variability and population diVerentiation. Three classiWcation methods were tested and compared: distance analysis, selforganizing map, and Bayesian methods, to evaluate the resolution of each method with ISSR markers. The 77 loci selected revealed a high variability between populations (polymorphism from 17% for Sian Ka'an to 75% for Banco Chinchorro) with a total polymorphism of 84% and a global coeYcient of gene diVerentiation (GST) of 0.296, but low values of Nei's Gene diversity (from 0.065 for Sian Ka'an to 0.233 for Banco Chinchorro). Our results suggest elevated inbreeding in all local populations with higher indices for Banco Chinchorro and lower indices for Sian Ka'an. Three independent classiWcation methods gave similar results, and suggested that most continental individuals are admixtures, with diVerent levels of introgression, with the sympatric species Crocodylus moreletii. We propose that the islands/atolls remain the sole areas with genetically "pure" American crocodiles and we discuss these results for future conservation of this endangered crocodile species. Inglés