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Aquilegia buergeriana var. oxysepala is a vulnerable herb, for which 20 microsatellite markers were developed. The applicability of these markers was confirmed by genotyping of 66 individuals collected from four natural populations in Japan. Of the 20 loci, 15 showed polymorphism. For the 15 polymorphic markers, the number of alleles per locus ranged from 2 to 12, and the expected and observed heterozygosities ranged from 0.13 to 0.77 and 0.02 to 0.65, respectively. The markers described here are sufficiently polymorphic and informative to investigate the genetic diversity and population genetic structure of A. buergeriana var. oxysepala.