A simple high-performance liquid chromatography (HPLC) method coupled with principal component analysis (PCA) and hierarchicalcluster analysis (HCA) was developed for distinguishing Astragali Radix from its adulterants. Five species, including A. membranaceusvar. mongolicus, A. membranaceus, H. polybotrys, A. chrysopterus and A. ernestii, were analyzed by this method. At the sametime, four major bioactive isoflavonoids, namely calycosin-7-O-D-glucopyranoside, ononin, calycosin and formononetin, were simultaneouslydetermined in Astragali Radix. This method was proven to have good precision, repeatability, stability and recovery. It wassuccessfully applied to distinguish Astragali Radix from its adulterants species and can be used for the quality control of Astragali Radix.