Premise of the study: Nuphar species (Nymphaeaceae) are representative aquatic plants in irrigation ponds in Japanese agricultural ecosystems. We developed 15 polymorphic microsatellite markers for N. japonica and confirmed their utility for its close relatives N. oguraensis var. akiensis and N. xsaijoensis, which originated from natural hybridization between N. japonica and N. oguraensis.Methods and Results: Genetic variation was characterized in 15 polymorphic loci in three populations of N. japonica. The average number of alleles per locus was 3.47 (range = 2-9; n = 32), and the average expected heterozygosity per locus was 0.84 (range = 0.5-1.0); 11 loci were amplified in N. oguraensis var. akiensis and 15 in N. xsaijoensis.Conclusions: The polymorphic microsatellite markers developed in this study will be useful for investigating the levels of genetic diversity within remnant populations of Nuphar taxa and could provide a valuable tool for conservation genetics of these taxa.