Background. Long-term lamivudine treatment induces the emergence of lamivudine-resistant hepatitis B virus (HBV). The objective of this study was to develop a fluorescent biprobe hybridization (FBH) assay for the detection and quantification of HBV mutants in the clinical course of lamivudine-treated patients and to evaluate its clinical usefulness. Methods. We developed an FBH assay to detect mutations in the HBV DNA polymerase gene. The assay's detection sensitivity was determined using a dilution series of wild-type/mutant plasmid DNA. Blood samples obtained from 27 lamivudine-treated patients were analyzed. Results. Mutant DNA levels as low as 10% of total HBV DNA were detected (sensitivity = 100%, specificity = 80%). HBV mutants were detected in five of the 27 patients during an average follow-up of 20 months after lamivudine administration. In one of the five patients, the YIDD mutant was detected at the initiation of lamivudine treatment, while the remaining four patients were identified as having YIDD mutants within 3 months after beginning lamivudine administration. Of the five patients with an HBV mutant, four developed breakthrough hepatitis more than 10 months after the detection of HBV mutants, following the reappearance or a re-increase of HBV DNA, characterized by a predominance of the mutant. The YIDD mutant was detected in one patient, even when the titer of the serum HBV DNA was below the detection limit of commercially available quantitative polymerase chain reaction. Conclusions. The FBH assay is an efficient method for detecting and quantifying HBV mutants, as early as 3 months after lamivudine administration.