Abstract: This study explored the genetic diversity and structure of 261 wild Rosa roxburghii Tratt. samples from 29 populations across eight provinces in China, utilizing 10 pairs of polymorphic EST-SSR primers and capillary electrophoresis, providing a clear genetic background for the collection and breeding of R. roxburghii germplasm resources. Results identified 95 alleles among the 261 germplasm materials. The average polymorphism information content of the loci was 0.568. At the population level, the average number of alleles and effective number of alleles were 3.131 and 2.331, respectively, with observed heterozygosity and expected heterozygosity averaging 0.508 and 0.488. The average Shannon’s information index was 0.858, indicating substantial genetic diversity, likely due to the extensive range of sampling sites. Analysis of population genetic differentiation revealed an average genetic differentiation coefficient of 0.067 and gene flow of 4.511, suggesting considerable inter-population gene flow. Analysis of molecular variance (AMOVA) indicated that most genetic variation in R. roxburghii came from within populations (r=0.467, P<0.000 1). Nei’s standard genetic distance among populations ranged from 0.054 to 1.269, with an average of 0.657, and was significantly related to geographical distance. Clustering analysis grouped the 29 populations into three main clusters, which was related to geographical location. These results highlight southwestern China as a core distribution area for wild R. roxburghii resources, providing theoretical guidance for the formulation of R. roxburghii conservation strategies.