Puerariae Radix was a widely used herbal medicine. Pueraria lobata (PL) and Pueraria thomsonii (PT) were the two authorized sources of Puerariae Radix (gegen) in China. In this study, metabolic differentiations between these two species were investigated using NMR spectroscopy followed by principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The content of puerarin in PL and PT was also determined usingquantitative H-1 NMR spectroscopy. Thirteen isoflavones were tentatively identified based on 1D and 2D NMR spectroscopic data in two species. The H-1NMR spectra of PL and PT were obviously different. PL and PT could also be markedly discriminated from H-1 NMR spectroscopic data by PCA and PLS-DA. For the crude drug resources, isoflavones, in which puerarin is the most important one, were regarded as the reasonable markers for the discrimination ofthe two species. The contents of puerarin and total isoflavones in PL were quantitated much higher than those in PT. Above all, H-1 NMR spectroscopy, which can provide comprehensive profiles of the metabolites and achieve convenient determinations of puerarin and total isoflavones in a single run, is an efficient means for evaluating the medicinal samples and achieving a better quality control of Puerariae Radix.