Traditionally, the authentication of the traditional Chinese medicines (TCM), Angelica sinensis, is based on slightly different morphological characters and complex compounds. Usually, those methods are simultaneously affected by several factors, leading to subtle and ambiguous results. In this study, the internal transcribed spacer (ITS) regions of A. sinensis and seven other Angelica species used as adulterants were sequenced. A pair of specific primers was designed from the polymorphic ITS regions to distinguish A. sinensis from the adulterants and regional substitutes. These ITS-derived primers amplified approximately 520 bp specific fragments from the adulterants, whereas no products was amplified with the DNA of A. sinensis. We tested eight commercially crude materials purchased in the market by using these specific primers. The result showed that there were four samples adulterating A. sinensis with regional substitutes. This indicated that A. sinensis could be accurately distinguished from the adulterants and regional substitutes. Therefore, the method of molecular authentication based on the ITS sequences may be contributed to raw material production and quality control of A. sinensis.