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The geographic origins of African slave settlers and the Portuguese genetic contribution to the population of São Tomé (Gulf of Guinea) were assessed through the analysis of b-globin haplotypes in 44 chromosomes bearing the bS allele and through the study of the genetic variation in eight autosomal markers (APOA1, AT3, FY, LPL, OCA2, RB1, Sb19.3, and GC) informative for admixture in a sample of 224 individuals. The observed bS haplotype distribution (36.4% Bantu, 52.3% Benin, 4.5% Cameroon, 4.5% Senegal, and 2.3% atypical) is in accordance with the historical information on the major geographic sources of slave settlers of São Tomé, although it captures a more important contribution of Central-West Africa regions than previously anticipated. European admixture, estimated to be 10.7 ± 0.9%, has created a considerable level of genetic structure, as indicated by the finding of significant linkage disequilibrium between 33% of unlinked marker loci pairs. Recent admixture was found to have an important contribution to these values, since removal of individuals with Portuguese or Cape Verdian parents or grandparents from the sample dropped the miscegenation level to 6.5 ± 0.8% and reduced significant linkage disequilibrium to 11% of unlinked marker pairs. Taken together, these results indicate that the peopling of São Tomé might have provided one of the first examples of the combination of diverse African contributions and European admixture that emerged from the overseas population relocations promoted by the Atlantic slave trade.