Using the HPLC/PDA/ESI/MS method, a comparative analysis of rhodionin (RH) was undertaken in order to conduct a qualitative and quantitative study in 38 batches of fourteen species of Rhodiola for quality control purposes. Alongside of this RH analysis, a simultaneous determination of salidroside (SA), tyrosol (TY), and gallic acid (GA) was carried out. Rhodiola plants are a popularly used ethnodrug from the Qinghai-Tibetan plateau of China. The identity of RH was unambiguously determined based on the quasimolecular ions in negative ESI-MS mode. This method was validated in respect to sensitivity, linearity, precision, repeatability and recovery using optimized chromatographic conditions. The linear calibration curve was acquired with R²>0.999, and the limit of detection (S/N=3) was estimated to be 43.75 μg/g. The relative standard deviations (RSDs) of the intra- and inter-day precisions were 0.75% and 0.50%, respectively. The repeatability was evaluated by a replicated analysis of samples with the RSD value found within 0.67%. The recovery rates varied within the range of 98.79—100.08% with RSD less than 1.10%. In the present study, the content of RH was quantified within 0.4192—4.7260 mg/g for 16 batches of R. crenulata. It was also found in eight other species plants. The results demonstrated that RH is a useful characteristic standard compound for quality evaluation and chemical differentiation among species of Rhodiola. The study also indicated that the analytical procedure is precise, reproducible and a potential tool for both quality assessment and species identification.