Certain biological mechanisms of Glehnia littoralis, an endangered medicinal plant in China, may be underlying its endangered status and this has attracted extensive attention from researchers. In this study, nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) sequences and random amplified polymorphic DNA (RAPD) were investigated to determine the genetic diversity of G. littoralis. A total of 291 samples from 11 G. littoralis populations were collected from the east coast of China. The high genetic diversity and large genetic differentiation within populations were reconfirmed by both ITS (Ht ¼ 0.576, Hs ¼ 0.512, G st ¼ 0.1526) and RAPD analyses (Ht ¼ 0.51, Hs ¼ 0.35, G st ¼ 0.31). The phylogenetic trees showed consistent topologies based on ITS and RAPD; all populations were divided into two clades. Mantel tests (ITS: r ¼ 0.3296, p ¼ 0.067; RAPD: r ¼ 0.036, p ¼ 0.418) showed no significant positive correlation between the geographical distance and genetic distance for G. littoralis populations. These results showed that G. littoralis has great evolutionary potential and its status as endangered might be related to special dispersal and germination mechanisms of its seeds, as well as habitat destruction. In conclusion, we suggest that the conservation strategy for G. littoralis should be based on the protection of all the wild populations to the extent possible.