Abstract: A method of high performance liquid chromatography tandem high resolution mass spectrometry (HPLC-HRMS) was developed for the separation and identification of chemical components in Rheum lhasaense A. J. Li et P. K. Hsiao extract. The separation of compounds was carried out on an Agilent Zorbax SB-C18 column (150 mm×4.6 mm×5 μm). The mobile phase was composed with acetonitrile and water with the flow rate of 0.5 mL/min. Electrospray ionisation source (ESI) was used, and Rheum lhasaense A. J. Li et P. K. Hsiao extract was analyzed by MS and MSⁿ full scan at positive and negative ion modes. Through comprehensive analysis of the mass spectra and chromatographic behaviors of the compounds, and comparing with the related data from standard substrates, literatures and database, the main chemical components of Rheum lhasaense A. J. Li et P. K. Hsiao extract were deduced and identified. A total of 63 compounds are identified from Rheum lhasaense A. J. Li et P. K. Hsiao extract at negative ion mode, and 54 compounds are identified at positive ionization mode. These compounds contain 22 tannins, 28 stilbenes, 6 flavonoids, 3 phenylpropanoids, 2 butyrophenones and 2 organic acids, which of 53 compounds are firstly discovered in Rheum lhasaense A. J. Li et P. K. Hsiao. And as reported in the literature, enquinones are not found. This method is fast and sensitive, which not only improves the identification efficiency of trace compounds in Rheum lhasaense A. J. Li et P. K. Hsiao complex matrix, but also achieves the analysis and identification of different types of compounds. It is helpful to provide scientific basis for further clarifying the material basis and quality control standards of Rheum lhasaense A. J. Li et P. K. Hsiao.