Major tropical rivers have been suggested to be important dispersal barriers that increase the beta diversity of animal communities in lowland rain forests. We tested this hypothesis using assemblages of frogs in the floodplains of the Sepik River, a major river system in Papua New Guinea. We surveyed frogs at five sites within a continuous 150 × 500-km area of lowland rain forest bisected by the Sepik, using standardized visual and auditory survey techniques. We documented 769 frogs from 44 species. The similarity in species composition decreased with logarithm of geographical distance between the sites, which ranged from 82 to 465 km. The similarity decay did not depend on whether or not the compared sites were separated by the Sepik River or whether the species were aquatic or terrestrial breeders. Likewise, a DCA ordination of frog assemblages did not show separation of sites by the river as a significant factor explaining their composition. Our results suggest that even major rivers, such as the Sepik, may not act as dispersal barriers. Rivers may not limit the distribution of frogs and therefore have a limited effect on determining frog species abundance and assemblage structure in rain forests.