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Rice (Oryza sativa L.) is the staple food of the majority of the population, particularly in Asia and Africa. Enriching rice with nutritional and therapeutic contents can improve its benefits for patients with lifestyle disorders. This study aimed to profile the phytochemical contents of the therapeutically known traditional rice Mappillai Samba against white rice CBMAS 14065 using non-targeted gas chromatography–mass spectrometry (GC-MS/MS). An analysis of the data using a mass spectrometry–data independent analysis (MS-DIAL) and MetaboAnalyst identified 113 metabolites belonging to 21 different classes of metabolites. A partial least square-discriminant analysis (PLS-DA) revealed 43 variable importance in projection (VIP) metabolites. This study identified therapeutically important metabolites, including phenylpropanoids, phytosterols, flavonoids, and polyamines, in the grains of Mappillai Samba. Three significant metabolic pathways, viz., phenylpropanoid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis, and steroid biosynthesis, were responsible for the grain metabolome variation between CBMAS 14065 and Mappillai Samba. Overall, the results of this study unravelled the biochemical complexity of Mappillai Samba, paving the way for the genetic mapping of the therapeutic compound accumulation in rice and the development of similar therapeutic rice varieties through molecular breeding.