Advanced third-stage larvae of Gnathostoma sp. corresponding to 6 morphotypes, distinguished on the basis of the shape of the cephalic hooklets, were collected from the muscle tissue of 5 species of freshwater fish (i.e., Dormitator latifrons, Eleotris picta, Gobiomorus maculatus, Ariopsis guatemalensis, and Cichlasoma trimaculatum) in Tres Palos Lagoon, Guerrero, Mexico. Principal components analysis of 4 morphological characters cluster all samples in a single taxonomic group. A minimum amount of variation was observed among ITS2 sequences of 3 morphotypes and Gnathostoma binucleatum obtained from GenBank (0–0.84%). The observed variation among morphotypes 1, 2, and 3 is the result of intraspecific variability of G. binucleatum supported by morphology and DNA. Morphotypes 4, 5, and 6 belong to the same taxon on the basis of morphology of the hooklets only. For an accurate morphological diagnosis of the causative agent of gnathostomiasis, it is necessary to develop similar studies with other species of the genus.