Summary The mutation of YMDD motif of hepatitis B virus (HBV) polymerase gene is the most frequent cause in HBV resistant to lamivudine. The aim of the study was to investigate variation features of HBV polymerase gene in chronic hepatitis B (CHB) patients before and after lamivudine treatment. From the serum samples of five CHB patients before and after 12 months of lamivudine treatment, HBV polymerase gene was amplificated and positive DNA fragments were cloned into JM105 competent cell. Twenty positive clones of every sample were checked with mismatched polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and YMDD variants were sequenced. Among five patients after 12 months of lamivudine treatment, M552I mutations in two patients with HBV DNA rebounding and D553G mutation in one non-responder were detected except two patients with negative HBV DNA consecutively. In summary, D553G mutation is probably one of the reasons that caused non-responders during lamivudine treatment. The mutations of YMDD motif occurred after lamivudine treatment are caused by the induction of lamivudine.