Stephania species is one of the alkaloid-rich genus of the family Menispermaceae. Most plants of the genus Stephania possess medicinal value, whose main components are alkaloids. However, the non-medical species are often mistakenly used as herbs because of the difficulty in identification of the species. A systematic method which involved the combination of DNA barcoding, HPLC-QTOF-MS/MS and UHPLC was established for differentiation, chemical profiles and quality evaluation of medicinal Stephania species. Firstly, twenty batches of Stephania species samples were classified into five Stephania species by DNA barcoding. Secondly, 114 alkaloids including 22 tetrahydroprotoberberines, 13 protoberberines, 27 aporphines, 13 benzylisoquinolines, 12 hasubanans, 3 morphines and 24 other alkaloids were clearly or tentatively identified. Thirdly, thirteen representative components were simultaneously detected by UHPLC-DAD to characterize the differences of chemical compositions among five Stephania species. In conclusion, this method was comprehensive and effective for identification, chemical profiles and quality evaluation of medicinal Stephania species. It will provide a basis for holistic quality evaluation of medicinal Stephania species.