The serologic heterogeneity of the hepatitis B virus (HBV) has been established from immunodiffusion experiments for a long time. Four serotypes called subtypes of the hepatitis B surface antigen (HBsAg) have been defined by two mutually exclusive determinant pairs, d/y and w/r, and a common determinant a. These subtypes are adw, ayw, adr and ayr. By subdivision of the four major subtypes in the mid-70s, nine different subtypes were identified. Sequencing of viral genomes has now become a major goal of descriptive virology, and sequence data is now used to trace routes of infection, to reconstruct the phylogenetic history of viruses and two delimit genetic subtypes. A genetic classification based on the comparison of complete genomes has defined four genomic groups of HBV, later referred upon as genotypes, which were designated with A-D. However, the interrelation of the nine subtypes to the genotypes, the possible presence of more than four human HBV genotypes as well as their global geographical prevalence remained to be determined. By sequencing the S-gene of HBV the molecular basis was assessed for the serological variations of HBsAg within the major four subtypes. Thereby, also two new genotypes of HBV designated with E and F were identified. Complete genomic sequencing of E and F strains confirmed their status as new genotypes. The F genotype was found to diverge from other HBV genomes sequenced by 14%, thus being the most divergent HBV genome so far characterized. When the worldwide molecular epidemiology of HBV based on the variability of the S-gene was defined, the E and F strains seemed to originate in aboriginal populations of Africa and the New World, respectively. They shared a unique substitution at residue 140 in the second immunodominant loop of their encoded surface antigen when compared to the vaccine strain. Future research will establish whether this substitution may predispose to a vaccine escape mutant at residue 141, that now has been reported to occur in conjunction with the 140 substitution.