Hepatitis B virus (HBV) genotype A has been divided recently into two subgroups, designated A-A' (genotype A excluding A') and A'. Isolates belonging to subgroup A' have been identified in Africa. A new genotyping method, based on PCR amplification of the pre-S/S genome region and subsequent restriction fragment length polymorphism (RFLP) analysis, was developed, that established a correlation between RFLP subtypes and subgroups within genotype A. To investigate the occurrence of subgroup A' in South America, 119 Brazilian HBV isolates were analyzed. Ninety-three (78%) of them belonged to genotype A, with three predominating RFLP subtypes: 44 (37%) isolates were classified as AI, 30 (25%) were AII, and 18 (15%) were AIII. Pre-S/S nucleotide sequences of 15 genotype A isolates were determined. Phylogenetic analysis performed with these 15 and an additional 41 sequences revealed that isolates AI and AII clustered in subgroup A', whereas isolates AIII were classified into subgroup A-A'. The correlation RFLP subtypes-subgroups was confirmed by the presence of amino acid residues specific for subgroup A' in the surface antigens and polymerase of isolates AI and AII. The high proportion (63%) of isolates from subgroup A' suggested an African origin for a large number of Brazilian HBVs.