Phellodendri Amurensis Cortex (PAC) is a medicinal herb that has been generally used to treat diarrhea and jaundice. In order to comprehensively evaluate the PAC in the main production areas quality, a qualitative and quantitative method with highly effective, sensitive, and reliable was developed. The chemical compositions of PAC were analyzed, and fingerprints were established by ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS). Then, the determination of berberine, canthin-6-one, dictamnine, γ-fagarine, and magnoflorine from PAC samples was simultaneously performed using UPLC-QQQ-MS. Furthermore, the chemical components of PAC from different regions were compared and analyzed by combining hierarchical cluster analysis, principal component analysis, and orthogonal partial least squares discriminant analysis. A total of 58 compounds were identified, including 36 alkaloids, four phenylpropanoids, seven terpenoids, four flavonoids and their glycosides, an organic acid compound, and six other components. The fingerprint results show that samples have good similarity. Meanwhile, the content of the five ingredients in different habitats is quite different. By multivariate statistical analysis, 18 batches of PAC could be divided into three categories, and 20 components were identified as differential markers of various origins. A comprehensive method of PAC quality evaluation and chemical composition difference analysis was established, which provided the scientific basis for quality evaluation and further pharmacological mechanism research.© 2024 Wiley-VCH GmbH.