The evolutionary history of eight-barbel loaches of the genus Lefua was investigated to resolve the faunal formation and evolutionary history of Japanese and East Asian freshwater fishes. In the present study, we found that seven Lefua specimens obtained from western Japan could not be assigned genetically to any population recognized so far, although they were morphologically and ecologically indistinguishable from Lefua sp 1. We tentatively designated the specimens as the Nihonkai population. Sequencing of the mitochondrial D-loop region showed that five specimens of the Nihonkai population were allied with the Kinki and Tokai populations of L. echigonia and other two with Lefua sp. 2, indicating a discrepancy between morphological-ecological and genetic traits. In order to determine whether the population experienced parallel evolution or introgression of mitochondrial DNA, we sequenced the nuclear ribosomal S7 subunit, and demonstrated that the Nihonkai population is included within Lefua sp. 1. These results indicate that the Nihonkai population can be identified as Lefua sp. 1, possessing introgressive mitochondrial DNA, and suggest that the evolutionary history and faunal formation of Japanese and East Asian freshwater fishes are more complicated than previously thought.