Background/Aims: The emergence of lamivudine-resistant hepatitis B virus (HBV) was reported in patients with prolonged lamivudine administration. There was no report of the existence of tyrosine–methionine–aspartate–aspartate (YMDD) mutant in non-lamivudine treated chronic hepatitis B patients. In the present study, we developed a sensitive assay and applied it to the detection of YMDD mutant. Methods: We developed peptide nucleic acid (PNA) mediated polymerase chain reaction clamping for detecting mutations in a YMDD motif of the hepatitis B virus DNA polymerase gene. We studied YMDD mutants in a patient with HBV DNA breakthrough longitudinally and in non-lamivudine treated patients (36 patients). Results: We could detect as little as 0.01–0.001% of mutant viruses coexisting in 10 5–10 9 copies of wild-type viruses using this assay. YMDD mutant was detected 7 months before clinical breakthrough, which was 6 months earlier than using the conventional restriction fragment length polymorphism assay. YMDD mutants were also detected in four of 18 anti-HBe antibody positive untreated chronic hepatitis type B: YMDD+tyrosine-valine-aspartate-aspartate (YVDD) in two patients and YMDD+tyrosine-isoleucine-aspartate-aspartate (YIDD) in two patients, however, none in HBe antigen positive patients. Conclusions: We developed a highly sensitive assay for detecting YMDD mutants. This is an effective procedure for monitoring patients during or before lamivudine treatment and can provide more insights into the therapeutic strategies for chronic hepatitis B patients.