Objective: To investigate the gene mutation patterns at hepatitis B virus (HBV) polymerase region in patients with lamivudine-resistant hepatitis B. Methods: The serum samples from 94 hepatitis B patients who underwent lamivudine treatment and developed resistance and another 50 hepatitis B patients without lamivudine resistance were collected. The resistant mutations at HBV polymerase gene region were detected by direct sequencing of PCR products. Results: Of the 94 lamivudine-resistant patients, 80 cases were found with lamivudine-resistant mutations at HBV polymerase gene region, among which the single mutation at RtM204I was 30.4% (24/80), and the alliance mutation of rtLl80M+rtM204V was 25.0% (20/80), rtLl80M + rtM204I was 22.5% (18/80) and rtVl73L + rtV180M + rtM204V was 8.3% (7/80), respectively. Only 2 cases of the 50 non-resistant patients had the HBV polymerase gene mutations. Conclusion: The lamivudine resistance-related mutations at HBV polymerase gene region are diverse and complex. Thus, multi-loci detection should be performed so as to find the drug resistance strains early and then adjust treatment plan accordingly.