Seven Y-chromosome STR loci, DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392 and DYS393 have been analysed in population samples of Angolares, Forros and Tongas, three ethnic groups from the African archipelago of São Tomé e Príncipe (Gulf of Guinea). Complete typings were obtained for 103 chromosomes, which belonged to 79 different haplotypes. The mean heterozygosity per locus in the overall São Tomean sample was 0.566, with the highest value found among Forros and the lowest among Angolares. Angolares also showed the lowest level of haplotype diversity. On average, the mean pairwise difference between two random haplotypes from Angolares, Forros and Tongas was 4.69, 6.74 and 6.23 repeats, respectively. The genetic distances were found to be statistically significant between Angolares and Forros or Tongas. In accordance, AMOVA revealed that the percentage of variation attributable to differences among groups was only significant when we distinguished between Angolares and non-Angolares. Globally, these results indicate that, with respect to the pool of male lineages of São Tomé e Príncipe, some genetic sub-structuring does exist, basically determined by the Angolares ethnic group.