Factors associated with hepatitis B virus (HBV) DNA breakthrough and the significance of YMDD variants without the presence of wild-type YMDD during prolonged lamivudine treatment are unknown. We studied the amino acid sequence of codon 552 (YMDD motif) and codon 528 by means of a line probe assay in 159 chronic HBV patients (median follow-up 29.6 months). Pretreatment HBV DNA levels and alanine transaminase (ALT) levels correlated inversely with the time to HBV DNA breakthrough with YMDD variants (r ؍ ؊0.46, P ؍ .001; r ؍ ؊0.45, P ؍ .001 respectively). Patients harboring YMDD variants 3 months before HBV DNA breakthroughs had higher HBV DNA breakthrough levels compared with those without YMDD variants 3 months before HBV DNA breakthroughs (18.9 ؋ 10 6 vs. 5.4 ؋ 10 6 copies/mL, P ؍ .007). Patients with HBV DNA breakthroughs had higher percentages of YMDD variants without the presence of wild-type YMDD compared with patients without HBV DNA breakthrough (25.6% vs. 9%, P ؍ .007 for single M552I variant; 20.9% vs. 8.1%, P ؍ .026 for single M552V variant; 30.2% vs. 9.9%, P ؍ .004 for M552I/M552V variants). Patients with HBV DNA levels of more than 10 3 copies/mL after 6 months of lamivudine therapy had a 63.2% chance of subsequently developing YMDD variants. HBeAg seroconversion occurred in 2 patients after the emergence of YMDD variants. Only one patient developed YMDD variant after HBeAg seroconversion. There was no increase in the rate of development of YMDD variants or L528M mutation in patients receiving lamivudine 25 mg daily or famciclovir 500 mg 3 times a day before being given lamivudine 100 mg daily. (HEPATOLOGY 2001;34:785-791.)Hepatitis B virus (HBV) infection is a major international health problem leading to around 1.2 million deaths per year world-wide. 1 Because of its profound suppression of HBV replication resulting in significant histologic improvement, lamivudine is commonly used as a treatment of chronic HBV infection. [2][3][4] However, in 14% to 32% of patients, a 1-year treatment regimen of lamivudine 100 mg daily is associated with mutation of the tyrosine, methionine, aspartate, aspartate (YMDD) motif in the C domain of the HBV DNA polymerase gene, corresponding to the amino acid codon 552 of the HBV. 2,3 The number of patients with YMDD mutation is higher with prolonged use of lamivudine. 5 For the YMDD variants, the methionine is substituted with either isoleucine (I), designated M552I or valine (V), giving rise to M552V. There may be another concomitant mutation occurring at the B domain of the HBV polymerase gene with the leucine (L) at amino acid codon 528 substituted by methionine (L528M). This mutation has been described for patients on famciclovir. 6,7 With lamivudine treatment, L528M is usually associated with M552V though M552I/L528M has also been reported. 8 Both in vitro and in vivo studies show that YMDD variants are less replication-competent compared with the wild-type, are associated with lower HBV DNA levels compared with pretreatment HBV DNA levels, and can...