Cytochrome P450 3A4 (CYP3A4) enzyme is involved in the metabolism of about 30 % of clinically used drugs, including the antimalarials artemether and lumefantrine. CYP3A4 polymorphisms yield enzymatic variants that contribute to inter-individual variation in drug metabolism. Here, we examined CYP3A4 polymorphisms in populations from malaria-endemic islands in Lake Victoria, Kenya, and Vanuatu, to expand on the limited data sets. We used archived dried blood spots collected from 142 Kenyan and 263 ni-Vanuatu adults during cross-sectional malaria surveys in 2013 and 2005-13, respectively, to detect CYP3A4 variation by polymerase chain reaction (PCR) and sequencing. In Kenya, we identified 14 CYP3A4 single nucleotide polymorphisms (SNPs), including the 4713G (CYP3A4∗1B; allele frequency 83.9 %) and 19382A (CYP3A4∗15; 0.7 %) variants that were previously linked to altered metabolism of antimalarials. In Vanuatu, we detected 15 SNPs, including the 4713A (CYP3A4∗1A; 88.6 %) and 25183C (CYP3A4∗18; 0.6 %) variants. Additionally, we detected a rare and novel SNP C4614T (0.8 %) in the 5' untranslated region. A higher proportion of CYP3A4 genetic variance was found among ni-Vanuatu populations (16 %) than among Lake Victoria Kenyan populations (8 %). Our work augments the scarce data sets and contributes to improved precision medicine approaches, particularly to anti-malarial chemotherapy, in East African and Pacific Islander populations.