A set of twelve EST-SSR markers were developed to evaluate the genetic diversity and genetic structure of Hemerocallis middendorffii Trautv. & C. A. Mey (Asphodelaceae) in Japan. The total number of alleles for each locus ranged from 3 to 19, with an average of 10.25; the mean values of H-O and H-E were 0.567 and 0.648, respectively. Versatility of the markers was evaluated for two additional species of the same genus, H. lilioasphodelus L. var. thunbergii (Baker) M. Hotta and H. hakuunensis Nakai to show that all the EST-SSR markers are transferrable between them.