In order to clarify the genetic background of the germplasm of Anemarrhena asphodeloides, the genetic diversity of 160 germplasm materials of A.asphodeloides was analyzed based on phenotypic traits and SSR markers. The results showed that the diversity index of 21 phenotypic traits ranged from 0.99 to 2.03, with an average value of 1.99. The coefficient of variation ranged from 17.22% to 151.07%, with an average of 43.42%. A total of 143 alleles were detected by capillary electrophoresis using 27 pairs of newly developed SSR primers. The effective alleles ranged from 0.56 to 7.40, with an average of 3.54. Shannon"s information index ranges from 0.17 to 0.51, with an average value of 0.36. Polymorphism information content (PIC) ranged from 0.15 to 0.50, with an average of 0.37. Cluster analysis showed that there was no obvious separation of 160 germplasm materials, but some of the same source of germplasm appeared in clusters. Principal component analysis and cluster analysis verified each other and showed similar results, which may be related to the frequent exchange of seeds and seedlings by the seed growers in different regions. In this study, 9 SSR markers associated with 6 phenotypic traits were screened by Chi-square test and T-test, which could be used for marker assisted breeding of female breeding.