Abstract: The seed of Peganumharmala Linn. belonging to the family of Zygophyllaceae, is a perennial herb which grows in semi-arid condition, steppe area and sandy soil. In China, Peganumharmala as a folk medicine has been used since antiquity among the Uighur, Kazakh and Mongolia for the treatment of cold, asthma, malaria, rheumatism, lumbago and some skin diseases such as scabies. The content of total alkaloids in ripe seeds could reach as high as 6% mainly in the form of harmaline and harmine, which were the first isolated in the last century. The problem was that the type and content of the rest of the alkaloids in the seeds of Peganumharmala were unknown. The aim of this work was to identify chemical constituents in the methanolic extract found in Peganumharmala. The extract of seeds of Peganumharmala were analyzed using high performance liquid chromatography coupled with high-resolution mass spectrometry. Chromatographic separation was achieved on a C18 column with the mobile phase of acetonitril-water (0.5% ammonia water) and step gradient elution. The ESI-MS analysis was operated at the range of m/z 100-1 000 in positive ionization mode. High-resolution mass spectrometry techniques have proven to be a reliable tool for the structural elucidation of unknown compounds in complex samples. In this context, linear ion trap quadrupole-Orbitrap-mass spectrometry (LTQ-Orbitrap MS) provides single-stage mass analysis that supplies molecular weight information and multi-stage mass analysis (MSⁿ) that provides structural information. Moreover, accurate mass measurement of the product ions facilitates the elucidation of unknown compounds. A total of twenty-two compounds of seeds are identified from the methanolic extract in the seeds of Peganumharmala, such as 10 isoquinoline alkaloid and 10 quinazolone alkaloids, which are characterized in the methanolic extract，based on the accurate mass of molecular and product ions provided by LTQ-Orbitrap mass spectrometer. As far as we know, these compounds have been recognized in seeds of Peganumharmala for the first time, such as glycosides vasicine, glycosides pegamine, 3-hydroxylated harmine. The method is rapid and simple in the qualitative analysis of licorice using LC-LTQ-Orbitrap MS, which could prove to have a high sensitivity and the result is accurate and reliable. It is an effective method to provide chemical information concerning the constituents in herbal medicines, which could be helpful for the quality control and further phytochemical studies of seeds of Peganumharmala L.