Rhodiola rosea, a high-altitude plant, is extensively being used in traditional folk medicine in Tibet to treat fatigue, asthma, haemorrhage, depression, anaemia, impotence, gastrointenstinal ailments, infections, and nervous system disorders [1]. Some other Rhodiola species, in particular, R. alterna, R. brevipetiolata, R. crenulata, R. kirilowii, and R. sachalinensis are often marketed as R. rosea, and thus, the therapeutic effects of R. rosea are not achieved. In an attempt to develop a mass spectrometry method for discriminating among these species, peptides from the six Rhodiola species were analyzed and isolated by high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-Tandem-MS). On the basis of the mass spectral fingerprints, 27 Rhodiola samples were successfully differentiated by principal component analysis (PCA) of the mass spectral raw data. The PCA results were also validated with cluster analysis and supervised PCA analysis. Using these fingerprints, some R. rosea-specific peptides were detected. This method provides effective and accurate identification of Rhodiola rosea.