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AIM: To develop a PCR assay using mutant-specific primers to detect mutation of tyrosine-methionineaspartate-aspartate (YMDD) motif of HBV to tyrosinevaline-aspartate-aspartate (YVDD) or tyrosine-isoleucineaspartate-aspartate (YIDD).METHODS: Cloned wild-type and mutant HBV sequences were used as templates to test the sensitivity and specificity of the assay. A variety of primer construction, primer concentration, dNTP concentration,and annealing temperature of primers were systematically examined. Pair primers specific to rtL180M and rtM204V were selected for YVDD detection. Primer specific to rtM204I with an additional 3'-penultimate base mismatched to both the mutant and wild-type sequence was selected for YIDD detection. We applied this assay to study YMDD mutants in 28 chronic hepatitis B patients before and after lamivudine treatment.RESULTS: We could detect as little as 0.001%-0.00001%of mutant viruses coexisting in 108-109 copies of wildtype HBV using this assay. YMDD mutants were detected in 8 of 12 HBeAg-positive patients and 8 of 16 HBeAgnegative patients before lamivudine treatment. After treatment, two more patients in HBeAg-positive patients and seven more patients in HBeAg-negative patients developed YMDD mutations.CONCLUSION: We developed a highly sensitive and specific assay for detecting YMDD mutants. This assay can be applied to monitor chronic hepatitis B patients before and during lamivudine treatment.