The genomes of six hepatitis B viral (HBV) strains were sequenced from 10 overlapping amplificates obtained by the polymerase chain reaction. Four of the strains, specifying subtypes ayw4 and adw4q-, represented on the basis of divergency within the S gene two new genomic groups identified by us. The other two strains, encoding adrq- and of Pacific origin, belonged to genomic group C. The relation of these genomes to 21 published human, 1 chimpanzee, and 4 rodent hepadnaviral genomes was analyzed by constructing a phylogenetic dendrogram. Thereby, the segregation of human HBV strains into six genomic groups was confirmed. A consistent grouping of the genomes compared was also obtained in dendrograms based on the P and S genes, although the branching order differed from that based on the entire genomes. Each of the two representatives of genomic groups E and F differed by 8.1 to 13.6% and by 12.8 to 15.5% from the genomes of the other groups and by 1.5 and 3.7% from each other. The two Pacific group C strains differed by 2.7% from each other and by 4.1 to 5.4% from other group C genomes, suggesting that they diverged early from the other group C genomes. The F strains formed the most divergent group of HBV genomes, which may be explained by their representing the original strains of the New World. Within the structural gene products, 17 and 34 amino acids unique for human HBV strains were recorded in the sequenced E and F strains, respectively. Most notable is the Ser81 to Ala81 substitution in an immunodominant region of HBcAg, and the four extra cysteine residues in HBsAg at residues 19, 183, 206, and 220, which might be engaged in additional disulphide bridges. Five residues shared by E and F strains were also unique for human HBV strains. Two of these, Leu127 and Ser140 in HBsAg, were the only substitutions that may explain the w4 reactivity shared by these HBV strains. Interestingly, the Ser140 substitution occurs in an immunodominant loop of the a determinant claimed to be important for the protective immune response to HBV vaccination.