This study was aimed at elucidating the common sequence variation present in the CYP2C19 gene within the South African Xhosa population and comparing it with the Cape Mixed Ancestry (CMA) population for possible future pharmacogenetic applications. Common sequence variation was identified through the resequencing of 15 Xhosa individuals. The detected variants were prioritized for genotyping in an additional 85 Xhosa and 75 CMA individuals, while 5´-upstream variants were analyzed using dual luciferase reporter assays. Resequencing of the Xhosa population revealed 30 variants, including the novel CYP2C19*27 and CYP2C19*28 alleles. CYP2C19*27, characterized by -1041G>A, caused a twofold decrease in luciferase activity, while CYP2C19*28 is characterized by the nonsynonymous V374I variant. In addition, the previously characterized variants, CYP2C19*2, CYP2C19*9 and CYP2C19*17, were present in both populations, while CYP2C19*3 was only observed in the CMA population. Our data demonstrate that both the Xhosa and CMA populations exhibit unique genetic profiles that could influence the outcome of drug therapy in these populations.