In this study, genetic diversity of Corydalis yanhusuo W.T.Wang was analyzed with SSR molecular markers, twelve pairs of primers with high polymorphism and good stability were screened for population genetic analysis for 360 individuals sampled from 19 populations. The results showed that: (1) a total of 227 polymorphic loci were amplified from 12 pairs of SSR primers, 4-9 alleles were detected and average of 5.25 per primer, which showed the primer pairs were of abundant polymorphism and could be used to analyze the genetic diversity of C. yanhusuo. The study revealed that the genetic diversity among the population groups was high (N_a = 5.25, I = 1.192 6, H = 0.387 9), the degree of genetic differentiation was high (F_st = 0.388 3), and the gene flow among species was limited (N_m = 0.393 8). (2) The results of cluster analysis used by both UPGMA and Bayes method showed that 19 populations obviously clustered into 4 branches. The result of Mantle test (r = 0.326, P = 0.01) indicated the population with shorter geographical distance performed tighter genetic relationship. The genetic structure of C. yanhusuo may be resulted from its predominantly selfing breeding system, clonal growth, geographic isolation and limited gene flow. In situ conservation should be accepted in its protection considering its genetic structure.