AbstractMedicinal herbs of high quality and with significant clinical effects have been designated as top-geoherbs in traditional Chinese medicine (TCM). However, the validity of this concept using genetic markers has not been widely tested. In this study, we investigated the genetic variation within the Rheum palmatum complex (rhubarb), an important herbal remedy in TCM, using a phylogeographic (six chloroplast DNA regions, five nuclear DNA regions, and 14 nuclear microsatellite loci) and a chemical approach (anthraquinone content). Genetic and chemical data identified two distinct groups in the 38 analysed populations from the R. palmatum complex which geographically coincide with the traditional top-geoherb and non-top-geoherb areas of rhubarb. Molecular dating suggests that the two groups diverged in the Quaternary c. 2.0 million years ago, a time of repeated climate changes and uplift of the Qinghai-Tibetan Plateau. Our results show that the ancient TCM concept of top-geoherb and non-top-geoherb areas corresponds to genetically and chemically differentiated groups in rhubarb.