During chronic HBV infections, exacerbations of disease usually occur without clearly understood mechanisms. In this study, full-length HBV genomes were analyzed from four chronic hepatitis B patients who developed resistance to lamivudine [(−)2′-deoxy-3′-thiacytidine, LMV] accompanied by acute exacerbation of disease. Paired full-length HBV isolates were cloned from the sera of patients prior to LMV treatment and after drug resistant breakthrough isolates emerged with exacerbation. Compared to the isolates before treatment, isolates from all four patients during exacerbation showed marked increase in replicative competence by cell transfection study. Viral genome amplification and direct sequencing was used further to study the sequence differences between the dominant species and the clones used for functional analysis. Apart from mutations at the YMDD motif, no shared mutations were shown between all isolates. The isolates from the one patient who recovered from the exacerbation showed a lower number of mutations, and in particular, lacked basal core promoter (BCP) mutations at 1762/1764. In contrast, BCP mutations were found in isolates from the other three patients. Thus, in patients with acute exacerbation, high replicative strains might be selected from the total HBV quasispecies during treatment, and amongst these strains, those with core promoter mutations were most likely to be associated with severe clinical exacerbations. J. Med. Virol. 77:203–208, 2005. © 2005 Wiley-Liss, Inc.