Panax notoginseng, a perennial herb belonging to the Panax genus, has a long growth and breeding cycle. The populations have low genetic diversity among populations but significant phenotypic variation within individuals of the population. Therefore, ^{P. notoginseng} is beneficial for the assessment of genetic diversity and the breeding of varieties by the development of molecular markers base on the whole-genome.In this study, simple-sequence repeat (SSR) markers were generated from the whole-genome and were applied to assess the genetic diversity among 11 populations (SL1-SL11) constructed based on saponin content. A total of 255,239 SSR markers were identified, and ultimately, 24 pairs of polymorphic primers were selected for genetic analysis. The results indicated that the heterozygosity of the 11 populations was similar (0.4583-0.6042), with low genetic differentiation (Fst =0.0447) and high gene flow (Nm = 11.6189). Total saponin content investigation revealed no difference among populations, but SL8 exhibited significantly higher than other populations in NG-Rg1. The trait association analysis indicated that SSR2 was associated with the content of G-Rg1, G-Re, and total saponin; SSR20 was associated with the content of NG-R1 and G-Rb1 content; SSR21 was associated with G-Rg1; and SSR22 was associated with NG-R1. In this study, SSR markers analysis were developed based on whole-genome information of P. notoginseng, which was helpful for population construction and variety breeding using molecular marker technology.