The molecular background of variant forms of GYPB is not well studied in Brazilians of African descent. The present study was carried out to determine the molecular bases of the S-s- phenotype and the frequency of GYPB*S silent gene for the S-s+ phenotype in a blood donor population of African Brazilians. In this study, 165 blood samples from African Brazilians (Northeastern Brazil) who phenotyped as S-s- (n = 17) and S-s+ (n = 148) by hemagglutination were selected. Allele-specific (AS)-PCR and PCR-restriction fragment length polymorphism (RFLP) were used to identify the variant forms of GYPB. In 13 of 17 S-s- samples (76.5%), both GYPB were deleted. In 137 of the 148 S-s+ samples (92.6%), the AS-PCR was consistent with the S-s+ phenotype. In 4 of the S-s- samples (23.5%) and 11 of the S-s+ samples (7.4%), the AS-PCR showed the presence of a GYPB*S allele associated with silencing of S. In the 4 donors with the S-s- phenotype, there was homozygosity (or hemizygosity) for the GYP(P2) allele (n = 2), homozygosity (or hemizygosity) for the GYP(NY) allele (n = 1), and heterozygosity for the GYP(P2) and GYP(NY) alleles (n = 1). In the 11 donors with the S-s+ phenotype, there was heterozygosity for GYP(P2) allele (n = 8) and heterozygosity for GYP(NY) allele (n = 3). This study reports for the first time the molecular mechanisms responsible for the S-s- phenotype in a population of African Brazilians and provides new information about the frequency and molecular bases of the GYPB*S silent gene (7.4%) in this population.