As a medicinal herb, Atractylodes macrocephala Koidz. experienced centuries of cultivation in China, and germplasm resources of all cultivated populations have degraded over time as a consequence of domestication processes. This study used chloroplast DNA and microsatellites to clarify not only the effects of domestication on population genetics, but also determine the geographic origins of landraces. The results revealed that cultivated populations (except the “Pingzhu” landrace) showed higher genetic diversity than their wild counterparts and low levels of genetic differentiation occurred between cultivated and wild groups. Furthermore, STRUCTURE and UPGMA analyses grouped all wild populations into three genotypic clusters, two of which (in Shaanxi and Hunan Province) shared the same gene pool with cultivated A. macrocephala, suggesting that wild populations in Central China have been involved in the origin of cultivated A. macrocephala. Moreover, the wild population from Qimen, Anhui Province and the cultivated “Pingzhu” landrace harbor unique gene pools and rare alleles that could be useful in future breeding efforts. This large-scale analysis of population genetics on a medicinal herb that has a centuries-long history of human-mediated selection will facilitate utilization and conservation of the valuable genetic resources of medicinal species.