Medicinal vines listed in Chinese pharmacopoeia possess important medicinal efficacy in traditional Chinese medicines.The ITS2 region, which has several characteristics that make it a valuable DNA barcode, was studied to discriminate the stems of medicinal vines to confirm their identities and ensure their safe application in pharmaceuticals by using complementary discrimination methods.Complementary discrimination methods were performed on two datasets, including 393 samples of 170 species from 22 genera 13 families, which belonged to medicinal vines and their adulterants. Based on the primary ITS2 sequences, three main discrimination methods (phylogenetic tree, the nearest distance, and BLAST 1) were adopted to identify species. Moreover, we applied both two-dimensional (2-D) and three-dimensional (3-D) structures of ITS2 to differentiate species.ITS2 performed well, with over 95.0% of species and 100% of genera being correctly differentiated for the two datasets. All results showed that the ITS2 region unveiled a remarkable ability to identify closely related species within different families and genera.Our findings supported that the ITS2 region was an efficient marker for authentication of medicinal vines.