Background: Hepatitis B virus (HBV) is replicated through reverse transcription of polymerase gene. Lamivudine can postpone the clinical progression in Hepatitis B infected patients, but the long-term monotherapy causes the emerge of YMDD (tyrosine-methionine-aspartateaspartate), LLAQ (leucine-leucine-alanine-glutamine) motifs and increases the Alanine aminotransferase (ALT) and HBV DNA levels. Objectives: The aim of this study was to investigate rtL180M and rtM204V mutations in polymerase gene of HBV in infected patients after Lamivudine therapy. Patients and Methods: Fifty sera samples collected from patients who referred to Blood Transfusion Center in Tehran were studied. The samples were divided into two groups; treated and untreated based on antiviral treatment status. From 50 patients, 34% were males and 66% were females, aged between 18 and 80 years. All of samples were tested for anti hepatitis B e antibody (anti-HBe), hepatitis B e antigen (HBeAg), hepatitis B surface antigen (HBsAg) and hepatitis B core antibody (anti-HBc) by enzyme-linked immunosorbent assay (ELISA) method. The ALT levels were measured using a commercial kit. Then HBV-DNA was extracted from serum samples using a commercial kit and a fragment of the P gene was amplified by nested PCR. Also, HBV viral load was detected by Real-Time PCR. HBV genotypes and polymerase gene mutations were determined by direct sequencing of the polymerase gene of HBV. Phylogenetic tree was constructed using neighbor-joining (NJ) method. Results: About 6% (3 of 50) of samples were HBeAg positive and 94% (47 of 50) of patients were HBeAg negative by ELISA method. The patients' ALT level was between 16 and 95 IU/L with the mean of 37.58 IU/L. Also, 48% (24 of 50) of samples had < 104 IU/mL viral load, 52% (26 of 50) of them had > 104 IU/mL viral load. About 10% (5 of 50) of samples was treated with Lamivudine with specific substitution of amino acid located at codons 80, 180 and 204. In addition, phylogenetic tree showed that genotype D of HBV was dominant among Iranian HBV infected patients Conclusions: This study showed that the presence of mutation at codons rtL80V, rtL180M and rtM204V in A, B and C domains is associated with higher viral load and resistance to Lamivudine (3TC) respectively.