Hepatitis B virus (HBV) is a global threat that killed many human lives. HBV DNA polymerase (HDP) is the key target for antiviral drug treatment. The widely used drug against HBV infection is lamivudine which targets the reverse transcriptase activity of HDP and inhibits the replication of HBV. However, available evidence demonstrated that tyrosine (Y)-methionine (M)-aspartic acid (D)-aspartic acid (D) motif mutations significantly affected the efficacy of lamivudine binding. In particular, M204I mutations affect the drug binding mechanism and cause resistance to lamivudine. Therefore, in the present study we made an attempt to understand the mechanism of lamivudine resistance with the aid of molecular docking and molecular dynamics (MD) approach. The molecular docking results suggest that lamivudine adopts the most promising conformations to the native type HDP by identifying M-204 and Y-203 as a prospective partner for making polar contacts as compared to the mutant type HDP. The MD results showed that the average movements of atoms, especially atoms of the native type HDP– lamivudine complex, were small and displayed fast convergence of energy and charges in geometry. This highlights the stable binding of lamivudine with native type HDP as compared to mutant type HDP. The R2 and RMSF analysis certainly indicates conformational changes in the HDP structure due to M204I mutation. Furthermore the hydrogen bond (H-bond) analysis from the MD study showed that there is decreased number of intermolecular H-bonds in mutant HDP – lamivudine complex as compared to that in native type HDP – lamivudine complex. Overall, our study certainly will pave way to develop new drugs against the drug resistant mutations (M204I) of HBV. © 2015, Springer Science+Business Media New York.