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The Japanese rose bitterling (Rhodeus ocellatus kurumeus) is facing imminent extinction because of hybridization and competition from an invasive alien subspecies (Rhodeus ocellatus ocellatus). Eleven new microsatellite markers for the two subspecies were developed using dinucleotide repeat specific polymerase chain reaction. The number of alleles per locus and the heterozygosity in R. o. kurumeus were lower than those in R. o. ocellatus. Most of these microsatellite markers were successfully cross-amplified in three Acheilognathinae species.