Two species Lefua echigonia and Lefua sp. 2 of the eight-barbel loach inhabit the Tokai region of Honshu, Japan. We determined sequences of the mitochondrial D-loop region to elucidate intraspecific phylogenetic relationships and variation in these two species. Lefua sp. 2 represented high intraspecific genetic similarity and complicated haplotype network, but three assemblages were recognized, including specimens mainly from Yahagi, Toyo, and Tenryu River systems, respectively, and named Groups 1 to 3. Divergence of Group 1 from the others was marginally supported, but Group 2 was paraphyletic to Group 3, suggesting the existence of two populations, i.e. Yahagi River population and Toyo-Tenryu River population. Lefua echigonia also represented high intraspecific genetic similarity, and two assemblages with slight genetic differentiation were discernible, including specimens from Shizuoka and southeastern Aichi prefectures and those from northwestern Aichi, Gifu, and Mie prefectures, respectively, and named Groups A and B. Star-like relationships of haplotypes suggested the dispersal origin located in eastern Aichi prefecture. The two species are threatened to extinction and thus we proposed evolutionary significant units for conservation.