We used DNA barcoding to identify the species of Pinellia and its adulterants, to authenticate medicinal plants of Pinellia. In this study, the matK region was amplified and sequenced for Pinellia ternata and its adulterants. The intraspecific and interspecific variation and barcoding gap were analyzed for ITS, ITS2, psbA-trnH, rbcL, matK region combined with GenBank database information, and the identification efficiency was evaluated based on Nearest Distance and BLAST1 methods. The results shown that matK region had the largest interspecific variation, and the rbcL region owned the smallest intraspecific variation. The rbcL region possessed the smallest overlap of intra- and interspecific genetic variation, followed by the matK region. Neighbor-joining tree of regions could divide species obviously. In conclusion, DNA barcoding could be used to accurately identify medicinal plants of Pinellia and its adulterants. The region of matK and rbcL was the ideal barcode combination for identifying the species of Pinellia and its adulterants.