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[Display omitted] • Metabolic profiles of 6 Dendrobium species were analyzed using 2D 1H–13C HSQC; • Characteristic metabolites of each of the 6 Dendrobium species were identified; • Common metabolites of Dendrobii herba were identified; • A protocol for high-throughput data analysis was developed and applied; Discrimination of the multiple botanical origins of herbal medicines is important to appropriately control their qualities and comprehensively understand their therapeutic effects. In our study, the chemical profiling of 6 different botanical origins of Dendrobii herba was analyzed using 2D 1H–13C HSQC. High-throughput data processing and analysis methods were developed and applied to detect and quantitatively characterize the spectral features of the 6 different Dendrobium species. Principal components analysis (PCA) and hierarchical cluster analysis (HCA) analyses of these spectral features showed a clear distinction among different Dendrobium species. Characteristic metabolite markers of each of the 6 Dendrobium species were identified by integrating the pairwise partial least-squares discriminant analysis (PLS-DA) and univariate hypothesis test. Besides, 8 major common metabolites were identified and found to have comparable content in all 6 Dendrobium species. Our results allow species authentication and differentiation of Dendrobium. It may also represent a universal protocol for authentication and quality assessment of medicinal herbs. [ABSTRACT FROM AUTHOR]