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Citrullus amarus(CA) (previously known as Citrullus lanatusvar. citroides) accessions collected in southern Africa are known to have resistance to root-knot nematodes (RKN) and are suitable rootstocks for grafted watermelon. The objective of this study was to conduct a comparative metabolomics analysis and identify unique metabolites in roots of CAaccessions versus roots of watermelon cultivars (Citrullus lanatus(Thunb.) Matsum. and Nakai var. lanatus; CL). Nuclear magnetic resonance (NMR) technology and principal component analysis (PCA) were used to analyze and compare metabolic profiles of seven CAaccessions resistant to RKN along with two RKN-susceptible watermelon cultivars (Charleston Gray and Crimson Sweet). Calculation of the Mahalanobis distance revealed that the CAUnited States Plant Introduction (PI) 189225 (Line number 1832) and PI 482324 (1849) have the most distinct metabolic profiles compared with the watermelon cultivars Charleston Gray and Crimson Sweet, respectively. Several amino acids identified in the CAaccessions were reported in previous studies to have a nematicidal effect. The results in this study indicate that roots of watermelon accessions collected in the wild are rich in metabolic compounds. These metabolic compounds may have been diminished in watermelon cultivars as a consequence of many years of cultivation and selection for desirable fruit qualities.