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Direct analysis in real time ionization source coupled with quadrupole orbitrap mass spectrometry (DART-Q-Orbitrap MS) was applied to analyze the Schisandra chinensis (S. chinensis) and Schisandra sphenanthera (S. sphenanthera) samples. The experimental condition including the ionization gas and gas temperature were optimized to obtain the best performance. The DART-MS analysis was operated using helium at 250 °C. The partial least squares discriminant analysis (PLS-DA) was conducted based on the DART-MS data to explore the differences between S. chinensis and S. sphenanthera samples. The clear separation between groups was observed in the PLS-DA score plot, indicating the chemome diversity of these two samples. Then 8 compounds that contribute most to the sample classification were selected and annotated, and the intensity change tendency of these compounds was similar to that obtained by the high-performance liquid chromatography (HPLC) method. Besides, these two species can also be discriminated by examining the existence of the compound anwulignan at m/z 328.1656 in this study. Our results show that DART-MS is a powerful analytical tool with the merit of rapid analysis speed, easy to handle, low consumption of organic solvent, and has the great potential for rapid detection and discrimination of S. chinensis and S. sphenanthera. It is expected that the established method could provide a rapid, reliable method for the quality assessment of Schisandra species, and expand this method to the analysis of other herbal medicines.