Abstract Background: Naturally occurred hepatitis B virus (HBV) with surface mutations in a variety of chronic hepatitis B (CHB) patients who have received no vaccine or HBIG bearing substitutions in surface protein, have been reported. Objectives: Current knowledge concerning the prevalence of these naturally occurring surface antigen mutations among Iranian carriers is limited. Patients and Methods: In a retrospective cross-sectional study, 119 inactive HBV chronic carriers were enrolled. The surface gene was amplified, sequenced and subsequently aligned using international and national sequence database. Results: All strains belonged to genotype D, subgenotype D1 and subtype ayw2. In 74 (62.18%) of patients, 146 (68.8%) out of 212 amino acid mutations occurred in different immune epitopes within surface protein, of which 28 (19.17%) in B cell, 37 (25.34%) in T helper and 81 (55.47%) inside CTL epitopes. 13 (8.9%) and 15 (10.27%) of amino acid substitutions occurred outside and within the “a” determinant in Major Hydrophilic Region (MHR). While 11 (9.24%) and 77 (64.7%) patients who harbored amino acid mutations, were HBeAg and anti-HBe positive, respectively (p=0.004). 9 and 63 amino acid mutations were occurred in different HBsAg epitopes in HBeAg and anti-HBe positive patients, respectively (P=0.04). Conclusions: HBV mutants within the surface immune epitopes seem to be extremely common among chronic carriers from Tehran, especially those who are anti-HBe positive, indicating that after HBeAg seroconversion, due to the selection pressure of e antibody, the occurrence of mutation is an inevitable effect of the evolutionary process.