This study aimed to identify the viral factors responsible for poor sensitivity to lamivudine (LAM). We analyzed 49 LAM-treated chronic hepatitis B patients infected with hepatitis B virus (HBV) genotype C. Serum HBV DNA reached a level below the detection limit of the sensitive PCR assay in 31 (63.3%) within the first 24 weeks of LAM therapy (good responder group). Of the patients who did not achieve undetectable levels of HBV DNA within 24 weeks (poor responder group), 15 (83.3%) experienced virological breakthrough, whilst only four patients in the good responder group (12.9%) experienced virological breakthrough. Multivariate analysis revealed that failure to achieve a reduction in viral load to undetectable levels within 24 weeks was independently associated with the occurrence of virological breakthrough. Sequence analysis of the HBV genome revealed that point mutations in the precore region (G1896A) and enhancer I (A1287G/C) were observed more frequently in the good responder group than in the poor responder group (P = 0.002 and 0.019 respectively), and the number of substitutions in the reverse transcriptase domain of the polymerase was significantly higher in the good responders than in the poor responders (P = 0.026). In conclusion, determining the sequence of preexisting HBV, especially for enhancer I, the precore region, and the RT domain of the polymerase region, may be useful in predicting sensitivity to LAM therapy.