Many species of Tetrastigma (Miq.) Planch. (Vitaceae) have long been used as medicinal plants in China, and some are endangered due to overexploitation. Although adulterants are often added to traditional Chinese medicines, there is no reliable or practical method for identifying them. In this study, we used four markers (rbcL, matK, trnH-psbA, and internal transcribed spacer [ITS]) as DNA barcodes to test their ability to distinguish species of Tetrastigma. The results indicated that the best barcode was ITS, which showed significant inter-specific genetic variability, and thus its potential as a DNA barcode for identifying Tetrastigma. Multiple loci provided a greater ability to distinguish species than single loci. We recommend using the combined rbcL+matK+ITS barcode for the genus. Phylogenetic trees from each barcode were compared. Analyses using the unweighted pair group method with arithmetic mean discriminated an equal or greater percentage of resolvable species than did neighbor joining, maximum likelihood, or maximum parsimony analyses. Additionally, five medicinal species of Tetrastigma, especially T. hemsleyanum, could be identified precisely using DNA barcoding.