Abstract: Many species of the genus Polygonatum have important medicinal value, but there is currently a lack of DNA barcodes that can effectively identify medicinal plants within Polygonatum. In the present study, based on the amplification and sequencing of ITS, trnK-matK, rbcL, psbA-trnH, and psbK-psbI, and the extraction of the ITS2 sequences, 138 sequences were obtained from 23 samples of seven Polygonatum medicinal species. Similarity search algorithm (BLAST) and neighbor-joining (NJ) analysis were used to compare the efficacy of sequences for identifying medicinal species and analysis of molecular variance (AMOVA) was applied to verify the reliability of the screened barcodes. Results demonstrated that the intra- and inter-specific genetic variation of trnK-matK showed less overlap and an obvious barcoding gap, while the other five sequences showed considerable overlap and no barcoding gaps. The BLAST search results showed that trnK-matK had the highest identification efficiency (85.7%). The phylogenetic trees also showed that trnK-matK had the most efficient identification ability, clustering all 12 samples of P. cyrtonema together and distinguishing P. sibiricum, P. kingianum, P. odoratum, P. punctatum, and P. zanlanscianense. AMOVA revealed that trnK-matK had the highest population genetic differentiation index (F_st) and was the most suitable for distinguishing differences between Polygonatum species. Therefore, among the six candidate barcodes, trnK-matK is the most suitable for molecular identification of medicinal plants of Polygonatum.