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To differentiate three medicinal Hippopahe species of seabuckthorn, a combined genetic and chemical identification method was established in this study. ITS2 and psbA-trnH were tested for identification of 3 species of seabuckthorn. Detection of the kimura 2-parameter (K2P) distance, the neighbor-joining (NJ) tree and the barcoding gap were used to assess the identification efficiency. ¹H-NMR based metabolic method was applied to acquire the profile of metabolites. PCA was used to analysis the metabolite data. The results indicated that DNA barcode combined ¹H-NMR based metabolic method is a powerful tool for the identification of 3 medicinal Hippopahe species of seabuckthorn. The finding demonstrated that different genetic variation and chemical constituents existed among 3 medicinal Hippopahe species of seabuckthorn. The combined identification method will improve the reliability of species discrimination and could be applicable to much other ethnic medicine which has various origins in China.