In order to reasonable utilize the medicinal plant resources and protect species diversity of Notopterygium incisum and Notopterygium franchetii, this project collected 227 individuals from 23 allpatric and parapatric populations of two Notopterygium species to detect the genetic diversity and species differentiation based on the simple sequence repeats (SSR). The results showed that: (1) N. incisum and N. franchetii had moderate level of genetic diversity. The average number of alleles (N_a), the number of effective alleles (N_e) and the expected heterozygosity (H_e) in N. incisum were 2.603, 1.777, 0.313, respectively. These diversity parameters were higher than that in N. franchetii, the values were 2.200, 1.641, 0.308, respectively. (2) AMOVA showed that the genetic variations were mainly occurred within populations for two Notopterygium species. The divergence index among populations F_st in N. incisum and N. franchetii, were 0.181 and 0.191, respectively. (3) Based on Bayesians structure clustering analysis and principal coordinates analysis, it was concluded that the all sampled individuals have been divided into two genetic clusters, which corresponded to N. incisum and N. franchetii, respectively. The obvious genetic differentiation and few gene exchanges between the two species were identified. These findings suggested that there were higher genetic differentiation among species, and their genetic variations mainly occurred within populations, the two geographic units for N. incisum and N. franchetii should be constructed for further management and conservation.