Abstract: This research used inter simple sequence repeat(ISSR)markers to analyze the genetic diversity of Hedyotis diffusa Willd. from different origins. A total of 23 samples of Hedyotis diffusa Willd. in the Guangxi Zhuang Autonomous Region, Guangdong, Hunan, Zhejiang, Fujian, Jiangxi and Anhui, respectively were collected. 150 ISSR primers were used to amplify PCR and then POPGENE1. 32, NTSYS2. 10 software were used to analyze genetic diversity. 11 primers were screened, 115 polymorphic bands were amplified, the polymorphism ratio was 85. 22%, the number of alleles(Na)was 1. 852 2, the effective allele(Ne)was 1. 543 4, Neis gene diversity index(H)was 0. 316 5 and Shannon′s information index(I)was 0. 470 0. The results of cluster analysis show that the Hedyotis diffusa can be divided into three clades. The conclusion is that ISSR molecular markers can provide a insight for the identification of Hedyotis diffusa Willd. .