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To assess the efficacy of sequential treatment with lamivudine and interferon-α monotherapies in Chinese patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B.One hundred and sixty-two patients with HBeAg-negative chronic hepatitis B were included in this study. Ninety-eight were treated with lamivudine alone (100 mg per day) for 48 weeks (group B). Sixty-four were treated with lamivudine alone (100 mg per day) for 20 weeks, then combined with interferon-α-2b (5 million units three times per week) for 4 weeks and then treated for another 24 weeks with interferon-α-2b alone (5 million units three times per week) (group A). All patients were followed for an additional 24 weeks.After 48 weeks of treatment, the percentage of patients with normalization of alanine aminotransferase (ALT) levels or hepatitis B virus (HBV) DNA levels below 1000 copies/mL was not significantly different between the lamivudine monotherapy group (55.10% and 55.10%, respectively) and the sequential treatment group (59.36% and 56.25%, respectively). The percentage of patients with normalized ALT levels was significantly higher in group A (53%) than in group B (36%) at week 72 (P < 0.05). The percentage of patients with lamivudine-resistant mutations was significantly higher with lamivudine monotherapy (22.45%) than with sequential therapy (P < 0.05).Sequential treatment of chronic hepatitis B with lamivudine and interferon-α monotherapies is as effective as lamivudine-alone treatment in Chinese patients. However, sequential treatment can significantly suppress the emergence of lamivudine-resistant mutations.