We used ISSR and RAPD molecular markers to analyze the genetic variation and relationship of 28 Paeoniae Radix Rubra (PRR), which provides a biological foundation for accurately evaluating the genetic characteristics, protecting the germplasm resources and cultivating the excellent new varieties of PRR. The results showed that: (1) 257 and 215 bands were amplified by 14 ISSR and 14 RAPD markers. The polymorphic bands and polymorphism ratio were found to be 251, 209 and 97.8%, 97.2%, respectively. Two molecular markers showed that the genetic diversity of wild PRR population was higher than that of cultivated population. (2) According to Shannons information index (I) and Neis gene diversity (H_e), the genetic diversity of population of Duolun County, Inner Mongolia (DL) was the highest, where can be recommended to establish a wild PRR nature reserve. (3) The genetic differentiation coefficient (G_st) indicated that the genetic differentiation of wild PRR populations was mainly within the population which may be caused by genetic drift. Conversely, the genetic differentiation of the cultivated PRR populations was mainly among the populations, indicating that there was less gene exchange among cultivated populations. (4) A UPGMA cluster grouped the 28 populations into 5 major clusters based on both ISSR and RAPD markers and their genetic distances were 0.115 1-0.343 8 and 0.095 5-0.286 2, respectively. This research shows that the complementary ISSR and RAPD methods can effectively analyze the genetic structure and genetic diversity of PRR populations at the DNA level.