Cypripedium tibeticum is a unique orchid species with high ornamental and medicinal value. As one endangered species, it is currently listed as a national protected plant. In this study, genetic structure and genetic diversity of 7 C. tibeticum populations collected in western China was analyzed using ISSR molecular markers. This study can provide a reference for species conservation in natural habitats of C. tibeticum. The results showed: (1) 12 polymorphic ISSR markers with strong repeatability were chosen from 100 ISSR primers. A total of 136 bands were identified, and the percentage of polymorphic bands (PPB) was 100%. Neis gene diversity (H_e) and Shannons information index (I) were 0.318 6 and 0.484 3, respectively. The Neis genetic distance ranged from 0.033 3 to 0.170 1 and genetic similarity ranged from 0.843 5 to 0.967 3 among populations. The overall gene differentiation coefficient (G_st) was 0.222 9 and the estimated value of gene flow was 1.743 0. (2) The UPGMA and NeighborJoining clustering analysis both indicated clear differentiation between populations from Sichuan and Shaanxi. The overall correlation between genetic distance and geographical distance was not significant. This study indicated high genetic diversity in C. tibeticum populations and great divergence between Sichuan and Shaanxi populations on molecular level. ISSR markers could be used for investigating the genetic structure, diversity and genetic differentiation of C. tibeticum populations.