The genus Profundulus is one of the oldest fish groups, distributed north of Central America in Mexico, Guatemala and Honduras. However, there are few studies that determine the number of species and their phylogenetic relationships. Historically it has been recognized that this taxon comprises two natural groups, defined as the subgenus Profundulus and Tlaloc. Recent genetic researches have revealed a high degree of differentiation between the two taxa and some species, probably associated with the morphological changes. In this way, we made the osteological comparison between six species of the subgenus Profundulus and Tlaloc (Cyprinodontidae: Profundulidae). We reviewed 17 bones located in the skull and pelvic girdle; qualitative differences were found in eight bones: the mesethmoid, vomer, lachrymal, premaxilla, articular, quadrate, interoperculum, and basipterygium. The variation patterns permit to diagnose both subgenus, corroborating the previous morphological and molecular studies. The evidence suggests that both taxa could be relocated as genus, such as previously was established.