Polygonatum cyrtonema Hua, a rhizome-propagating herb endemic to China, is often used in many traditional Chinese medicines and foods. The hilly mountains in western and southern Anhui province is one of its main natural distribution and artificial cultivation areas. The genetic diversity and structure of P. cyrtonema germplasm resources in Anhui were assessed by 9 pairs of SSR primers. The results showed that the 13 sampled populations of P. cyrtonema possessed normal levels of genetic diversity and could be clustered into three genetic groups, i.e. group I (MC, FLS, BJS, HHGD, JHGS, JHHS), group II (YLC, HG, HGGS, HT), and group III (TTZ, YLP, TZS). The levels of genetic diversity of three groups were similar, but their distribution areas and morphological characters were remarkably different. Group I is confined to Tianmu (including Jiuhua) Mountains, Group II is distributed in Huangshan Mountains, and Group III is restricted in Dabie Mountains. In morphology, the leaf length:width ratio significantly differed among groups, and the peduncle length of group I was significantly shorter than that of the other two groups. In addition, the genetic differentiation among groups was closer to that of related species within the genus. Thus these three genetic groups of P. cyrtonema should be considered as independent units for conservation and breeding management in the Anhui region.