Salvia miltiorrhiza is an outcrossing and perennial herb native to China. Although well-known for its medicinal value, there is a lack of knowledge regarding its natural population genetics. Here, we used 12 microsatellite markers to investigated population genetic diversity and structure of 215 samples from populations naturally distributed in central eastern China. A moderate level of genetic diversity was detected probably due to the over-mining of its roots. The allelic richness (AR) ranged from 3.034 to 4.889 with an average of 3.891. Moreover, pairwise estimates of FST among the populations of S. miltiorrhiza varied from 0.036 to 0.312 and two clusters were obtained by STRUCTURE and discriminant analysis of principal components. It is likely that the genetic differentiation of these two clusters was formed during glacial periods. Our result provides insights into the conservation of this valuable medicinal plant.