Mitochondrial and nuclear DNA analyses were used to examine the native distribution range and population structure of Biwia zezera (Cyprinidae: Gobioninae) based on specimens from the species' presumed distribution range. We found two greatly differentiated groups, with 8.6% uncorrected sequence differences in the mtDNA cytochrome b gene; one group was distributed exclusively in the Yodo River system (excluding Lake Biwa and rivers flowing into the lake). This differentiation was supported by results from three nuclear DNA loci, and it was concluded that the Yodo population is an undescribed cryptic species. Based on mtDNA haplotype distribution and endemicity, B. zezera was determined to be indigenous to the Ise Bay area (Nobi Plain), Lake Biwa basin (excluding the Yodo River outlet and its tributaries), the Sanyo region, and northern Kyushu. The population in the Ise Bay area was the most differentiated from the others, while that in Kyushu was the second-most differentiated. It was confirmed that an endemic population closely related to the Lake Biwa population is naturally distributed in the Sanyo region.