Twenty microsatellite loci were isolated from a hybrid of two daylilies, Hemerocallis fulva and Hemerocallis citrina. We characterized individuals from two H. fulva populations and two H. citrina populations in Japan and observed three to 20 alleles per locus in H. fulva and one to 19 alleles per locus in H. citrina. Mean observed heterozygosity within populations ranged from 0.35 to 0.85 in H. fulva and from 0 to 0.95 in H. citrina. In about a half of the loci, the observed heterozygosity did not deviate from Hardy–Weinberg equilibrium. These loci are proved useful in studying gene flow and qualitative trait loci mapping using the two species.