Lamivudine therapy often causes breakthrough of hepatitis B virus (HBV) DNA and breakthrough hepatitis. The aim of this study was to determine the viral factors that relate to HBV-DNA breakthrough with and without breakthrough hepatitis. Among 82 patients with chronic hepatitis B (CHB) who received lamivudine at a dose of 100 mg daily for more than 24 months, 23 patients had HBV-DNA breakthrough induced by a lamivudine-resistant mutant. Of these 23 patients, 16 had breakthrough hepatitis and 7 had only HBV-DNA breakthrough. Serial HBV-DNA full-genome sequences during therapy were examined in 10 (7 had breakthrough hepatitis and 3 did not) of these 23 patients by direct sequencing. Mutations in the S region were examined by cloning in representative patients. There were no significant differences in the baseline clinical backgrounds and virus marker between patients with and without breakthrough hepatitis. The HBV amino acid substitutions at breakthrough hepatitis were identical to those at HBV-DNA breakthrough. Cloning analysis revealed that monoclonal mutational strain appeared at breakthrough and no such mutations existed at baseline. Regarding HBV amino acid substitutions in the polymerase region, S region, X region, and precore-core region with breakthrough compared to baseline, there was no significant differences of the numbers of amino acid substitution between breakthrough hepatitis and non-breakthrough hepatitis. There were no common amino acid changes in patients with breakthrough hepatitis. Although monoclonal lamivudine-resistant strain emerged at HBV-DNA breakthrough in patients with CHB, there were no common amino acid changes, suggesting viral factor may have insignificant role in breakthrough hepatitis. J. Med. Virol. 79:911–918, 2007. © 2007 Wiley-Liss, Inc.