The genus Polygonatum, collectively known as Polygonatum or Solomon’s Seal, contains a number of species that have edible rhizomes that are used in traditional Chinese medicine. Technology to trace the origin of harvested plant materials is crucial. Our study selected five indicators: stable isotope δ 13C, δ 15N, δ 2H, δ 18O, and polygona-polysaccharose in Polygonatum rhizome to distinguish the origin of samples from six cities in southern China, with a cross-validation rate of 100%. In addition, we studied the correlation between the content of δ 13C, δ 15N, δ 2H, δ 18O, and polygona-polysaccharose in Polygonatum samples and environmental parameters. We found a significant positive correlation between δ 18O and latitude, and a significant positive correlation between δ 2H and longitude and annual evaporation. Based on this, we believe that the combination of the four stable isotopes and polygona-polysaccharose of Polygonatum can serve as indicators for tracing the origin of rhizomes.