From several gnathostome species the complete internal transcribed spacer ITS-2 ribosomal DNA (rDNA) repeat sequence and a fragment of the 5.8S rDNA were obtained by direct polymerase chain reaction cycle-sequencing and silver-staining methods. The size of the complete ITS-1 sequence in agarose gel electrophoresis was also obtained. The ITS-2 enabled the differentiation of Gnathostoma spinigerum from Thailand and Gnathostoma binucleatum from Mexico and Ecuador and confirmed the validity of the latter. Gnathostoma turgidum, Gnathostoma sp. I (=Gnathostoma procyonis sensu Almeyda-Artigas et al., 1994), and Gnathostoma sp. II (=G. turgidum sensu Foster, 1939 pro parte), all from Mexico, proved to be independent species, but Gnathostoma sp. III, also from Mexico, could not be differentiated from G. turgidum. In Mexico and Ecuador, gnathostomes involved in human infection and that had been classified as G. spinigerum belong to G. binucleatum. The 5.8S rDNA sequences of the 6 Gnathostoma species studied were identical. The results of the ITS-1 agreed with those results of ITS-2.