Antillean manatees (Trichechus manatus manatus), a subspecies of the West Indian manatee, is listed as endangered species in the Red List of Threatened Species of the International Union for Conservation of Nature. The aims of this research were to survey on the possible regional genetic structure in the southern Gulf of Mexico and to compare genetic status of a landlocked population in Laguna de las Ilusiones (IL) with individuals from localities with no barriers to displacement and breed (open population [OP]). We analyzed 45 manatee skin samples collected from different locations in Tabasco (n = 38, including 19 from IL), Veracruz (n = 3), Campeche (n = 2), and Chiapas (n = 2). The genomic DNA was isolated and PCR amplifications were performed for each sample using 28 microsatellite loci, previously designed for West Indian manatees and described as polymorphic for this species. Two clusters (k = 2) were identified by STRUCTURE. The analysis of both a priori populations (IL and OP) indicate that the global values of F_ST and R_ST (F_ST=0.049, R_ST=0.077) were significant. The H_E for IL was 0.38 ± 0.03 and for OP was 0.49 ± 0.01. The average number of alleles N_A for IL was 2.21 ± 0.09 and for OP was 2.32 ± 0.09. The overall inbreeding coefficient was F_IS=−0.013 for analyzed populations. Genetic diversity was low. The IL population had slightly lower genetic diversity compared with OP, which could be explained by isolation of that small group, so conservation plans for IL should be considered as priority.