Abstract The three living species of manatees, West Indian (Trichechus manatus), Amazonian (Trichechus inunguis) and West African (Trichechus senegalensis), are distributed across the shallow tropical and subtropical waters of America and the western coast of Africa. We have sequenced the mitochondrial DNA control region in 330 Trichechus to compare their phylogeographic patterns. In T. manatus we observed a marked population structure with the identification of three haplotype clusters showing a distinct spatial distribution. A geographic barrier represented by the continuity of the Lesser Antilles to Trinidad Island, near the mouth of the Orinoco River in Venezuela, appears to have restricted the gene flow historically in T. manatus. However, for T. inunguis we observed a single expanding population cluster, with a high diversity of very closely related haplotypes. A marked geographic population structure is likely present in T. senegalensis with at least two distinct clusters. Phylogenetic analyses with the mtDNA cytochrome b gene suggest a clade of the marine Trichechus species, with T. inunguis as the most basal trichechid. This is in agreement with previous morphological analyses. Mitochondrial DNA, autosomal microsatellites and cytogenetic analyses revealed the presence of hybrids between the T. manatus and T. inunguis species at the mouth of the Amazon River in Brazil, extending to the Guyanas and probably as far as the mouth of the Orinoco River. Future conservation strategies should consider the distinct population structure of manatee species, as well as the historical barriers to gene flow and the likely occurrence of interspecific hybridization.