ISSR molecular markers were employed to assess the genetic diversity and differentiation of Anoectochilus roxburghii and it’s relative species collected from different districts of China. Results indicated that, a total of 156 bands were amplified from 50 samples by 9 ISSR primers, and 155 were polymorphic bands, the percentage of polymorphic bands was 99.36%; UPMGA dendrogram analysis indicated that, the similarity coefficient ranged from 0.56 to 0.90, at genetic similarity of 0.65, all the 50 samples were clustered into 4 groups, and A.roxburghii were mainly gathered in group A. Group A can further clustered into 3 subgroups and 5 small subgroups, and there were no significantly boundaries among A.roxburghii samples sourced from different districts. After removed unknown source samples, based on geographic source, 46 samples were divided into 8 populations, included 4 Fujian wild A.roxburghii populations, the values of Nei’s gene diversity (H) of them were 0.322 and 0.295, which were in average level compared with other orchids; the coefficient of genetic differentiation among population(Gst) were 0.328 and 0.240, which were in low level compared with other orchids.Results of AMOVA Analysis indicated that genetic variation within population was 87%, variation among population was 13%, indicated there could exist gene flow among populations.