Lamivudine is an effective antiviral agent for the treatment of chronic type B hepatitis. Recent studies have shown the appearance of lamivudine resistant viruses with mutations at the tyrosine-methionine-aspartate-aspartate (YMDD) motif of the viral polymerase in hepatitis B virus (HBV) infected patients who received orthotopic liver transplantation. In order to confirm the appearance of such mutant HBV in immunocompetent patients, the HBV sequences in and around the YMDD motif of HBV DNA polymerase were examined in the sera from 16 lamivudine treated and 10 untreated control patients. Approximately 200 bases including the YMDD motif of HBV DNA polymerase were amplified by polymerase chain reaction (PCR) and sequenced directly by an automated sequencer. Of the 16 patients receiving lamivudine, mutant viruses with mutations in the YMDD motif were found in 3 of 8 patients treated with lamivudine for 52 weeks. However, this mutation was not found in any of the 8 patients treated for 32 weeks or a shorter period. Mutant viruses appeared after 40 weeks of treatment and were undetectable within 12 weeks after the cessation of the treatment. Such mutant viruses were not detected in any of the 10 untreated patients. This study confirms the emergence of YMDD mutant viruses during long-term lamivudine treatment in immunocompetent type B hepatitis patients. The results from this study suggest the need for combination therapies to reduce the levels of such mutant viruses in some patients. J. Med. Virol. 60:8–16, 2000. © 2000 Wiley-Liss, Inc.