The genetic diversity and genetic structure of 6 populations of Panax notoginseng and its two related species were analyzed by using 17 pairs of EST-SSR molecular markers in this research. As a result, a total of 205 EST-SSR polymorphic loci were detected in 8 populations. The PIC (Polymorphism information content) of six cultivated populations of P. notoginseng was 0.729, Gene diversity within population (H) was 0.1568,Shannon’s information index (I) was 0.2466, and the coefficient of gene differentiation (Gst) was 0.2350 among populations of P. notoginseng. The results revealed abundant genetic diversity but low level of genetic differentiation in P. notoginseng and confirmed that the genetic difference of the species existed mainly within population. In addition，Genetic similarity and cluster analysis showed that 8 populations of P. notoginseng and its related species were classified into three groups, 6 populations of P. notoginseng were classified into three subgroups. Genetic distance was closer between P. notoginseng and P. japonicus var. major than between P. notoginseng and P. stipuleanatus