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As herbal medicines have an important position in health care systems worldwide, their current assessment and quality control are a major bottleneck.Cortex Phellodendri Chinensis and Cortex Phellodendri Amurensis are widely used, however how to identify species and control the quality of CPA and Cortex Phellodendri Chinensis has become urgent.In this study, ultra-performance liquid chromatography/electrospray-ionization quadruple-time-of-flight high-definition mass spectrometry and multivariate statistical analysis were performed.Principal component analysis showed that two herbs could be separated clearly.The chemical markers such as berberine, plamtine,phellodendrine, magnoflorine, obacunone, and obaculactone were indentified through the S-plot of orthogonal partial least squared discriminant analysis, and were identified tentatively by the accurate mass of quadruple-time-of-flight mass spectrometry and partially verified by the available reference standards.A total of 29 components can be used as the chemical markers for discrimination of Cortex Phellodendri Amurensis and Cortex Phellodendri Chinensis.Of them, phellodenrine is significantly higher in Cortex Phellodendri Chinensis than that of Cortex Phellodendri Amurensis, whereas obacunone and obaculactone are significantly higher in Cortex Phellodendri Amurensis than that of Cortex Phellodendri Chinensis.The present study proves that ultra-performance liquid chromatography-mass spectrometry based chemical analysis greatly contributes to the investigation of Cortex Phellodendri Amurensis and Cortex Phellodendri Chinensis, and showed that the identified chemical markers as a whole should be used to discriminate the two herbal medicines, and simultaneously the results also provided chemical information for their quality assessment.