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Objective: Belamcandae Rhizoma and Iridis Tectori Rhizoma are easily confused with each other. The main objective of this study is to distinguish them using chemical analysis. Materials and Methods: Thin?layer chromatography (TLC) and high?performance liquid chromatography (HPLC) fingerprint methods were established to compare the chemical profile, while HPLC quantitation was used to determine the contents of three isoflavones in thirty batches of Belamcandae Rhizoma and Iridis Tectori Rhizoma samples. Results: The two herbs could be distinguished by TLC using acetic acid?n hexane?ethyl acetate (1:90:80 v/v/v) as the mobile phase, according to the fluorescent band under 366 nm at Rf 0.2. In total, 12 compounds were identified in the 24?min HPLC fingerprint. The similarity coefficient between the two herbs was 0.54 ± 0.01. Mangiferin (1), tectoridin (2), iridin (3), irigenin (5), irisflorentin (6), and iristectorin A (9) were the main peaks in Belamcandae Rhizoma, while tectoridin (2) and tectorigenin (4) were the major peaks in Iridis Tectori Rhizoma. The contents of 2 in Iridis Tectori Rhizoma (2.50 ± 0.20 ％) were 8.93 times higher than that of Belamcandae Rhizoma (0.28 ± 0.08 ％), while the ones of 5 and 6 were slightly lower in Iridis Tectori Rhizoma. Conclusions: The study established fast and effective methods to distinguish Belamcandae Rhizoma from Iridis Tectori Rhizoma.