Molecular epidemiology of hepatitis B virus (HBV) often relies on the comparison of HBV surface (S) gene sequences, although little is known about the substitution rate of the HBV S-gene. In this study, we compared HBV S-gene sequences in longitudinal sample pairs of 40 untreated, chronically HBV-infected patients, spanning 210 years of cumulative follow-up. The 40 patients included HBV e-antigen positive and negative persons; with HBV DNA levels ranging from 10(3) to 10(9) cps/mL and belonging to HBV genotypes A, B, C, D and E. In the 40 sample pairs, 70 nucleotide changes occurred in the HBV S-gene (0-8 per patient), resulting in an average substitution rate of 5.1 x 10(-4) nucleotide changes/site/year (range: 0-1.3 x 10(-2)). Surprisingly, the number of substitutions was strongly associated with the inverse level of viremia; and only weakly with the duration of follow-up: in 11 highly viremic patients (HBV DNA > or =10(8) cps/mL), only four substitutions occurred despite a cumulative observation period of 56 years (substitution rate: 1.1 x 10(-4)), while in the 10 patients with viremia below 10(4) cps/mL, 29 substitutions occurred during 30 years of follow-up (substitution rate: 14.6 x 10(-4)). We conclude that in chronic hepatitis B virus infection the rate of nucleotide substitution in the HBV S-gene is inversely related to the level of viremia and thus varies widely from person to person; hampering the phylogenetic analysis of possible chains of HBV infection.