We have analyzed the molecular dynamics of emergence of drug-resistant strains in patients receiving lamivudine therapy for chronic hepatitis B. Twenty consecutive patients with lamivudine resistance were studied (13 hepatitis B e antigen [HBeAg]–positive patients and 7 HBe antibody [anti-HBe]–positive patients). Determination of viral genotype, precore mutants, and polymerase gene mutants (L528M, M552V, M552I) was performed using the research version of Lipa-HBV. Quantitative analysis of HBV DNA was performed using both branched DNA (bDNA) and polymerase chain reaction (PCR) assays. Polymerase mutants (genotypic resistance) were found in 16 of 20 patients. Genotypic resistance was detected earlier than the phenotypic resistance (P = .004). Quantitative PCR allowed detection of viral DNA throughout the entire study period in 16 of 20 patients. Analysis of pretreatment variables showed that high alanine transaminase (ALT) levels (>3 × the upper limit of normal [ULN]) was associated with a more rapid selection of drug-resistant mutants (P = .027) and a high hepatitis B virus (HBV) DNA level (>1,497 Meq/mL, bDNA) with a more rapid occurrence of phenotypic resistance (P = .04). At the time of viral breakthrough, the mean serum HBV-DNA values were not different from the pretreatment values (P = .37). ALT levels were higher in anti-HBe–positive patients compared with pretreatment values and to HBeAg-positive patients (P = .01). In 8 patients, antiviral therapy was modified after viral breakthrough, with the introduction of famciclovir and/or interferon alfa. Viral DNA became undetectable by bDNA in 3 patients who received interferon. Our results suggest that genotypic assays for polymerase mutant detection and quantitative determination of viremia with highly sensitive assay are warranted for an optimal monitoring of antiviral therapy of chronic hepatitis B.