Rhodeus atremius is an endemic bitterling fish from Japan comprising two endangered subspecies, R. a. atremius and R. a. suigensis. The latter subspecies, which had dramatically declined in last decades, was reported to contain two distinct mtDNA lineages. In order to estimate the optimized units for conservation management, the genetic structure of R. atremius was inferred by analyzing mtDNA and microsatellite markers (MS). Allelic richness and heterozygosity of MS in R. a. suigensis was less than half that in R. a. atremius. In R. a. suigensis, not only within-population genetic diversity but also among-population genetic divergence was low, with the exception of population Ah1, while the diversity was high and the divergence showed isolation by distance in R. a. atremius. In mtDNA and MS, R. a. suigensis concordantly formed a single lineage, while R. a. atremius encompassed four mtDNA lineages, two of which were completely admixed into one group on the basis of MS. In population Ah1 a striking introgression between the two subspecies was suggested by a Bayesian-based assignment test, with the presence of mtDNA haplotype of R. a. atremius. Contrary to the prevailing theory, R. a. suigensis corresponds to a single conservation unit, while three units seem appropriate for R. a. atremius. In addition, low genetic diversity of R. a. suigensis might have arisen mainly as a result of recent bottlenecks before population fragmentation, followed by current anthropogenic effects. Genetic introgression in population Ah1 was probably the result of human transplantation of R. a. atremius.