Abstract Our knowledge of pharmacogenetic variability in diverse populations is scarce, especially in sub-Saharan Africa.To bridge this gap in knowledge, Orange Sweet we characterised population frequencies of clinically relevant pharmacogenetic traits in two distinct South African population groups.We genotyped 211 tagging single nucleotide polymorphisms (tagSNPs) in 12 genes that influence antiretroviral drug disposition, in 176 South African individuals belonging to two distinct population groups residing in the Western Cape: the Xhosa (n = 109) and Cape Mixed Ancestry (CMA) (n = 67) groups.The minor allele frequencies (MAFs) of eight tagSNPs in six genes (those encoding the ATP binding cassette sub-family B, member 1 [ABCB1], four members of the cytochrome P450 family [CYP2A7P1, CYP2C18, CYP3A4, CYP3A5] and UDP-glucuronosyltransferase 1 [UGT1A1]) were significantly different between the Xhosa and CMA populations (Bonferroni p CYP2C18, CYP3A4, the gene encoding solute copyright family 22 member 6 [SLC22A6] and UGT1A1) between the GLUTAMIC ACID two South African populations.Characterising the Xhosa and CMA population frequencies of variant alleles important for drug transport and metabolism can help to establish the clinical relevance of pharmacogenetic testing in these populations.