Hedysarum polybotrys is commonly substituted for Astragalus membranaceus in Taiwan for many years. This study described several different ways, including microscopic morphology, ITS (internal transcribed spacer) sequencing and HPLC fingerprint methods to identify these two species. Furthermore, the antioxidant activities of the two plants were evaluated and H. polybotrys had higher antioxidant activity than A. membranaceus. All the methods gave consistent conclusions that test samples KRA2, 3 and 8 belonged to A. membranaceus, while KRA1, 4, 5, 6 and 7 belonged to H. polybotrys. The results revealed that all the above methods may successfully discriminate between A. membranaceus and H. polybotrys.