Harpagophytum procumbens (Pedaliaceae) and its close taxonomical ally H. zeyheri, indigenous to southern Africa, are being harvested for exportation to Europe where phytomedicines are developed to treat inflammation-related disorders. The phytochemical variation within and between natural populations of H. procumbens (n=241) and H. zeyheri (n=107) was explored using proton nuclear magnetic resonance (1H-NMR) and ultra high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in combination with multivariate data analysis methods. The UHPLC-MS results revealed significant variation in the harpagoside content: H. procumbens (0.17 - 4.37%); H. zeyheri (0.00 - 3.07%). Only 41% of the H. procumbens samples and 17% of the H. zeyheri samples met the pharmacopoeial specification of 1.2%. Both principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) indicated separation based on species (UHPLC-MS data OPLS-DA model statistics: R2X = 0.258, R2Y (cum) = 0.957 and Q2 (cum) = 0.934; 1H-NMR data OPLS-DA model statistics: R2X = 0.830, R2Y = 0.865 (cum) and Q2 (cum) = 0.829). It was concluded that the two species are not chemically equivalent and should not be used interchangeably.