Abstract: The ISSR-PCR marker was used to study relationships and genetic polymorphism for nine species and one variation of Hemerocallis. Ten primers were screened from 123 random primers. A total of 218 DNA fragments were amplified. They were polymorphic. The molecular evidence indicated that different species in the genus were high polymorphism. Nei’similarity coefficients and genetic distances was calculated by TFPGA. The dendrogram of nine species and one variation in the genus can be constructed by UPGMA. The results indicated that the nine species and one variation of the Hemerocallis can be divided into four groups. H. lilioasphode, H. citrina, H. fulva, H. dumortieri, H. fulva var. kwanso were in the first group; The second group was the cluster of H. forrestii, H. plicata and H. minor. The third group was H. flava, the fourth group was H. nana. The genetic distance among the different species ranged from 0.0187 to 0.4941. The closest relationship was H. lilioasphode and H. citrina. The next was H. forrestii with H. plicata and H. minor. H. fulva, H. dumortieri, H. fulva var. kwanso were coming after that. H. nana kept the further genetic distance with other daylily species.