The traditional medicinal plant, and endangered species Aristolochia delavayi (Aristolochiaceae) is an endemic species in China and occurs in the warm and dry areas along the Jinsha river. It is also a specific host of the larvae of Byasa daemonius, a vulnerable butterfly. In this study, 15 pairs of polymorphic microsatellite primers of A. delavayi were designed and screened based on the Simple Sequence Repeats (SSR) loci found by using the results of genome skimming. Based on these 15 SSR markers, the genetic diversity and structure of 193 individuals from ten natural populations were analyzed in detail. In comparison to other endemic and endangered plants in the region, the population of A. delavayi possess a relatively high genetic diversity (He=0.550, I=1.112). AMOVA analysis showed that 68.4％of the total genetic diversity was within populations and 31.6％of the variation occurred among populations. There was a significant genetic differentiation among natural populations of A. delavayi detectable, with low gene flow (Nm=0.591). This might be attributed to geographical barriers and limited seed dispersal. To test the isolation by distance (IBD), we performed Mantel test, which showed a significant correlation between the geographic and genetic distances. In order to cope with the possible biases caused by IBD, we additionally performed Bayesian genetic cluster analyses and principal coordinate analysis (PCoA). The final cluster analysis revealed three groups with distinct geographical distribution. Habitat frag-mentation and limited gene flow between these populations may be the main reasons for the current genetic structure. For conservation of this species, we suggest to divide its populations into three pro-tection management units, with subsequent focus on the Yongsheng and Luquan populations which experienced a genetic bottleneck event in the past.