Abstract: In this study, sixteen samples of Gymnadenia conopsea were collected, and the fingerprints of Gymnadenia conopsea were established using ¹H-NMR metabonomics technology. Combined with principal component analysis (PCA) and other chemometrics methods, the differences of primary and secondary metabolites of Gymnadenia conopsea from different regions were systematically analyzed at metabolome level, and the quality of Gymnadenia conopsea from different regions was entirely evaluated. By comparison, the results indicated that GC-1, GC-2, GC-3, GC-4, GC-5, GC-6, GC-7, GC-8, GC-9 and GC-10 had higher quality, and the established ¹H-NMR spectrum provided a new technical method for the quality control and evaluation of Gymnadenia conopsea. The results provide reference for the cultivation, breeding, quality control development and utilization of Gymnadenia conopsea.