Treatment of hepatitis B virus (HBV) with lamivudine is effective in suppressing virus replication and results in reduced inflammatory activity. However, the emergence of lamivudine‐resistant mutant virus, with amino acid substitution in the YMDD motif of DNA polymerase, has been reported. We report the emergence and takeover of YMDD mutant and re‐takeover by wild type during and after long‐term lamivudine therapy. YMDD mutants were detected in five patients who showed DNA breakthrough (HBV DNA becoming detectable after a period of DNA negativity), which occurred after 9 to 14 months of lamivudine therapy. Four of five mutants had amino acid sequence YIDD, and the remaining mutant had YVDD. Patients with high HBV‐DNA titer and/or hepatitis B e antigen tended to develop breakthrough (P = .038). Using a sensitive and specific polymerase chain reaction (PCR)‐based method developed in this study, the emergence of YMDD mutants was detected 1 to 4 months before DNA breakthrough, but not detected in any of the pretreatment sera. The mutants were predominant at breakthrough, but were replaced by wild‐type virus 3 to 4 months after cessation of therapy in the two patients who discontinued therapy. One of these patients had a relapse of hepatitis. Mutant continued to replicate in the remaining three patients who continued to receive treatment, and relapse occurred in only one of these patients. Our results suggest that the replication of YMDD mutant viruses is less than wild type and is re‐overtaken by wild type after cessation of therapy. Re‐administration of lamivudine, possibly combined with other antiviral therapy, might be useful in some patients experiencing hepatitis with lamivudine‐resistant variants.