Abstract: Semen Lepidii is the dried and mature seeds of the cruciferous plant of Descurainia Sophia or Lepidium Apetalum, being a widely-used traditional Chinese medicine for various pharmacological effects, such as antibacterial, antitumor, diuretic and improving immune function. However, the chemical components of Semen Ledidii from different regions have not been clear, limiting the further application of this herb medicine in clinic. Therefore, in this study, a method of ultra-performance liquid chromatography-electrostatic Orbital ion trap tandem mass spectrometry (UPLC-Orbitrap-MS/MS) was developed to identify the chemical components of the three Semen Lepidii, namely Descurainia Sophia 1 (DS1), Descurainia Sophia 2 (DS2) and Lepidium Apetalum (LA) from different regions, and to study the fragmentation pathways of the identified components. The methanol-water (70:30, V/V) extract of the three mashed powder samples was separated on an ACQUITY UPLC BEH C18 column (100 mm×2.1 mm×1.7 μm), and infused into high mass resolution Orbitrap mass spectrometer for analyzing under positive and negative ion modes. MS/MS data were acquired with a Full MS/dd-MS² mode, and the chromatographic peaks were aligned and extracted by using Compound Discoverer (CD) software. Then, the accurate mass informations of the precursor and fragment ions were matched to the databases in the CD platform for identification of components in the samples, and studied the fragmentation pathways of identified compounds. A total of 39 compounds are identified in DS1, including alkaloids, flavonoids, cardiac glycosides, amides, organic acids, amino acids, and others. This method was used to analyze methanol-water extract of DS2 and LA, 37 compounds are identified in DS2, including 10 compounds which are not identified in DS1, and 42 compounds are identified in LA, including 7 compounds which are not identified in both DS1 and DS2. The results indicated that the chemical components of Semen Lepidii originated from different regions are remarkably different and may act as medicine in various ways. This study provides a fundamental for further studying the molecular mechanism of pharmacological effects of Semen Lepdii, which can promote its clinical application.