The pretherapy factors that could influence the emergence of resistant hepatitis B virus (HBV) tyrosine-methionine-aspartate-aspartate (YMDD) motif mutants against lamivudine are not fully known in prolonged lamivudine therapy for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B. We analyzed prospectively 116 consecutive lamivudine-naïve patients who received long-term lamivudine therapy (>1 year) by using multivariate regression analyses. The cumulative HBeAg loss rates were 29, 44, and 47% at 1, 2, and 3 years of treatment, respectively. Stepwise Cox's regression analyses indicated that pretherapy viral load was a significant factor associated with HBeAg loss (P = 0.0068). The cumulative emergence rates of YMDD mutants were 23% at 1 year, 45% at 2 year, and 47% at 3 year of treatment. Stepwise Cox's regression analyses indicated that patient age and presence or absence of severe acute exacerbation of liver disease were independent significant factors associated with emergence of YMDD mutants (P = 0.018 and 0.048, respectively). For the development of virological breakthrough, patient age, the presence or absence of severe acute exacerbation, and pretherapy viral load were independent significant factors (P = 0.028, 0.043, and 0.044, respectively). Severe acute exacerbation tended to reduce or delay development of biochemical breakthrough. The present study provides important information for the development of more effective and rational long-term lamivudine therapy for HBeAg-positive chronic hepatitis B patients infected exclusively with genotype C. J. Med. Virol. 73:7–12, 2004. © 2004 Wiley-Liss, Inc.