The dried root tubers of Stemona tuberosa, S. japonica and S. sessilifolia are the original sources of Stemonae Radix (SR) for antitussive and insecticidal activities. The products of SR which are available on the market are variable, and imitations exist. In order to characterize the overall chemical constituents of SR and evaluate its quality, a novel, binary high-performance liquid chromatographic fingerprinting method, describing the pattern of alkaloids (fingerprint I) and non-alkaloids (fingerprint II) of SR was developed. It was also applied to determine whether the medicinal parts and the processing methods affect the quality of SR. Similarity and high-performance liquid chromatography-mass spectrometry (HPLC-MS(n)) were utilized to compare or identify the chemical constituents of SR. The results indicate that the chemical constituents from different parts of the underground material of Stemona plants are diverse and that the processing methods affect certain constituents in the root tuber samples. The similarity and the resulting chemical consitituents obtained show that the binary chromatographic fingerprint method can be used to differentiate the three official Stemona species or the adulterants of SR, which is helpful for the identification and quality evaluation of SR.