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Sea buckthorn (Hippophaë rhamnoides L.) is well-known for its high content of flavonol glycosides (FGs), yet accurately determining the quantity of these nutrients present in the berries remains a challenge due to the significant variability of these nutrients and lack of reference standards. In this study, flavonol glycosides in sea buckthorn berries were identified and quantified using ultra-performance liquid chromatography Q-exactive high resolution mass spectrometry (UPLC-QE HRMS). The results showed that the total content of flavonol glycosides ranged from 39.5 to 240.2 mg/100 g fresh berries. The wild berries from Sichuan had remarkably high contents and unique profiles of flavonol glycosides. The main flavonol glycosides in all of the samples were isorhamnetin glycosides and quercetin glycosides, the predominant flavonol glycosides were different between different samples. The sPLS-DA statistical analysis indicated that cultivated and wild sea buckthorn can be distinguished based on their distinct composition and content of flavonol glycosides. The HCA clustered 16 high expression levels of FGs, which were revealed in a wild sample obtained from Sichuan. • High resolution and high sensitivity analysis was carried out on UPLC-QE HRMS. • 33 flavonol glycosides identified and quantified by UPLC-QE HRMS in sea buckthorn. • Differences of the flavonol glycosides between cultivated and wild samples were analyzed. • sPLS-DA analysis revealed clear distinction between cultivated and wild sea buckthorn. • High expression flavonol glycosides identified by hierarchical cluster analysis. [ABSTRACT FROM AUTHOR]