Genetic structure and its artificial disturbance in the endangered Japanese bitterling Acheilognathus cyanostigma were examined based on mitochondrial cytochrome b gene sequences. The haplotype network included three major clades: the dominant clade consisted of haplotypes found from the Lake Biwa–Yodo River system and other sites, including a known site of introduction; the other two clades consisted of haplotypes found exclusively or almost exclusively from the Ise Bay area, and the Yura and Kako River systems, respectively. Their divergence times, estimated using teleost molecular clocks, yielded results concordant with geological events such as uplifting of the Suzuka Mountains during the early Pleistocene. The widespread haplotypes were suggested to be introduced from the Lake Biwa–Yodo River system during stocking of Ayu from this system. The Lake Biwa–Yodo River population is critically endangered in its original habitats, yet is invasive in habitats where it has been introduced as it hybridizes with or replaces indigenous populations and competes with native bitterlings. Utilizing introduced populations for reintroduction and genetic rescue of the original population may be possible after careful examination for factors that could negatively impact the local community.