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Cupressus gigantea W. C. Cheng & L. K. Fu is an endemic conifer tree species that is distributed widely along the northern portion of the deep gorge of the Yarlung Tsangbo River on the Tibetan Plateau. However, as a key plant species growing on the Tibetan plateau, C. gigantea has since become an endangered species due to habitat loss and degradation, overexploitation, and other factors. It has been listed as a first-grade national protected wild plant species in China. Accordingly, to conserve this plant species, we should obtain more information on its genetic structure. In this study, the genetic diversity and structure among 67 samples were evaluated by the inter-simple sequence repeat (ISSR) technique. Overall, 78 bands were produced with a molecular length of 200 bp to 3100 bp using 10 ISSR primers. The mean values for the average number of alleles (Na), effective number of alleles (Ne), Nei's gene diversity (H), and Shannon's information index (I) were 1.529, 1.348, 0.199, and 0.293, respectively. Additionally, the number of polymorphic loci (NPLs) and percentage of polymorphic loci (PPLs) averaged 41.25 and 52.90, respectively. Further, total variation among populations was 14.2%, while that within populations was 85.8%; accordingly, the within-population genetic differentiation was found to be significant (p < 0.001). These results demonstrated that a genetic structure model with K = 3 fitted the data best, which agreed with the unweighted pair group method with arithmetic average (UPGMA) cluster and the principal coordinate analysis (PCoA). These findings are beneficial for ensuring the development and genetic protection of C. gigantea populations in the future. [ABSTRACT FROM AUTHOR]