Abstract: Physalis Calyx seu Fructus, a traditional Chinese medicine consisting of the calyxes and fruits of Physalis alkekengi L var.franchetii (Mast.) Makino has been used as therapy for inflammation-related respiratory diseases such as excessive phlegm, cough, sore throat, and pharyngitis for a long history in China. It is widely distributed in Asia and Europe, and mainly grows in Yunnan, Sichuan, Guizhou, Shanxi, and Henan Provinces of China. The calyx and fruit of Physalis Calyx seu Fructus possess a variety of properties, including anti-inflammatory, antimicrobial, antitumor, and anti-diabetes. The chemical constituents of the dried fruit and calyx extract of Physalis Calyx seu Fructus were analyzed and identified by ultra-performance liquid chromatography-Q-exactive Orbitrap high resolution mass spectrometry (UPLC Orbitrap HRMS) method in order to further understand its chemical composition. Thermo Syncronis C18 column was used for sample separation, 0.1% formic acid solution (A)-acetonitrile (B) was used as mobile phase for gradient elution. The electron spray ionization (ESI) ion source and Orbitrap mass analyzer were equipped, and negative ion mode was used for data collection. Based on the accuracy of relative molecular mass, fragment ions, retention behavior compared to the database, a total of 23 components were identified, including 5 organic acids, 12 flavonoids, and 6 physalins. Kaempferide-3-O-glucoside and malvidin-3-O-glucoside were first discovered in Physalis Calyx seu Fructus. This method can provide a method reference for the study of mass spectrometry fragmentation pathways of similar compounds, and also can provide a material basis and lay the foundation for the future application.