Astragalus radix is a well-known herbal material in traditional Chinese medicine. Astragalus membranaceous and A. membranaceous var. mongholicus are known as the authentic origins of Astragalus medicines whereas Hedysarum polybotrys is a common adulterant of Astragalus radix in Taiwan. Random amplified polymorphic DNA (RAPD) primers were used to screen the polymorphic fragments. Among them, OPB-2 and OPC-14 directed the amplification of 3 specific fragments A1 (1.3 Kb), A2 (0.6 Kb) and H1 (0.8 Kb) from A. membranaceous, A. membranaceous var. mongholicus and H. polybotrys respectively, which were further converted to sequence characterized amplified region (SCAR) markers. Primer pairs HG3 and HG4 derived from A1, HG7 and HG8 from A2, and RC3 and RC4 from H3, were successfully employed on the authentication of Astragalus medicines and H. polybotrys.