Abstract: Polygonatum Mill. (Asparagaceae) is a pharmaceutically important genus with many species are of significant medicinal value. Taxonomy and interspecific identification of Polygonatum species have long been controversial due to their considerable morphological variation, wide geographic distribution, complex speciation processes, and lacking of high-resolution molecular markers. To evaluate species discrimination power of 14 plastid divergence hotspot regions (candidate sequences) and their combinations in Polygonatum, a total of 166 individuals from 32 populations representing 15 medicinal Polygonatum species distributed in China were sampled for study. The interspecific and intraspecific genetic variation of each sequence and sequence combination were estimated, and tree-based and pairwise genetic distance (PWG-distance) methods were applied. The results indicated that except for trnT-trnL, the designed primers for all the other 13 candidate sequences showed good universality. Varying degrees of overlaps were detected between intraspecific and interspecific genetic distances in each of the 14 single candidate sequences and their combinations. Nonetheless, overlaps in the combined sequences were significantly lower than those in single sequences. Species resolution of the 14 single sequences were 6.67%-40% and 20%-60% based on tree-based and PWG-distance methods, separately. The combined sequences possessed higher species-resolving power with 40%-73.33% by tree-based method and 46.67%-73.33% by PWG-distance method, accordingly. Among them, the combined sequences C0 and C1 (in both tree-based and PWG-distance methods), C2 and C3 (in tree-based method), and C25 (in PWG-distance method) all showed the best resolution degree of 73.33%, indicating that combination of sequences could effectively improve species discrimination power. In addition, sequences psaJ-rpl33, rps16-trnQ, trnF-ndhJ, trnT-trnL, trnK-matK and atpF all exhibited relatively higher species-resolving degree, which could be used as specific molecular markers for the identification of medicinal Polygonatum species, and we propose the combination of psaJ-rpl33+rps16-trnQ+trnF-ndhJ+trnK-matK+atpF as the most ideal high-resolution molecular marker for discriminating the medicinal Polygonatum. This study will provide a basis for conservation and utilization of germplasm resources and accurate identification of medicinal Polygonatum, as well as standardizing the market for Polygonati Rhizoma.