Hepatitis B virus is classically considered a hepatotropic virus but also infects peripheral blood mononuclear cells. Chronic hepatitis B has different disease phases modulated by host immunity. We compared HBV variability, drug resistance and immune escape mutations in the overlapping HBV polymerase/surface gene in plasma and peripheral blood mononuclear cells in different disease phases. Plasma and peripheral blood mononuclear cells were isolated from 22 treatment naïve patient cohorts (five inactive, six immune-active, nine HBeAg negative and two immune-tolerant). HBV was genotyped via line probe assay, hepatitis B surface antigen titres were determined by an in-house immunoassay, and HBV DNA was quantified by kinetic PCR. The HBV polymerase/surface region, including full genome in some, was PCR-amplified and cloned, and ˜20 clones/sample were sequenced. The sequences were subjected to various mutational and phylogenetic analyses. Clonal sequencing showed that only three of 22 patients had identical HBV genotype profiles in both sites. In immune-active chronic hepatitis B, viral diversity in plasma was higher compared with peripheral blood mononuclear cells. Mutations at residues, in a minority of clones, associated with drug resistance, and/or immune escape were found in both compartments but were more common in plasma. Immune escape mutations were more often observed in the peripheral blood mononuclear cells of immune-active CHB carriers, compared with other disease phases. During all CHB disease phases, differences exist between HBV variants found in peripheral blood mononuclear cells and plasma. Moreover, these data indicate that HBV evolution occurs in a compartment and disease phase-specific fashion.