Human and animal nanophyetiasis is caused by intestinal flukes belonging to the genus Nanophyetus distributed on both North American and Eurasian coasts of Northern Pacific. In spite of the wide geographical distribution and medical and veterinary importance of these flukes, the intra-generic taxonomy of Nanophyetus spp. remains unresolved. The two most widely distributed nominal species, Nanophyetus salmincola and Nanophyetus schikhobalowi, both parasitizing humans and carnivorous mammals, were described from North America and eastern Eurasia, respectively. However, due to their high morphological similarity their interrelationships remained unclear and taxonomic status unstable. In this study, we explored genetic diversity of Nanophyetus spp. from the Southern Russian Far East in comparison with that of samples from North America based on the sequence variation of the nuclear ribosomal gene family (18S, internal transcribed spacers, ITS1-5·8S-ITS2 and 28S). High levels of genetic divergence in each rDNA region (nucleotide substitutions, indels, alterations in the secondary structures of the ITS1 and ITS2 transcripts) as well as results of phylogenetic analysis provided strong support for the status of N. salmincola and N. schikhobalowi as independent species.