Background: Mutations in the S gene (HBsAg), precore (PC) and basic core promoter (BCP) of the hepatitis B virus (HBV) are correlated with a wide spectrum of diseases. This study assessed the frequency of mutations in the S gene, PC and BCP regions in chronic hepatitis B (CHB) patients. Methods: 104 CHB patients who visited Tehran Hepatitis centers, were included. The viral load of samples was determined based on the TaqMan method. Regions of the S gene, PC and BCP were amplified by the nested PCR. Positive PCR products were sequenced and analyzed. Results: Successfully sequenced S gene region revealed all the derived strains were genotype D, with the majority (90%) belonging to the ayw2 subtype, and the rest (9%) to the ayw1 subtype. The prevalence of mutations was found to be 51% and 18% in the HBsAg and MHR regions, respectively. 70% of amino acid changes within HBsAg occurred in different immune epitopes, of which 27% and 72% were located in B cell and Th epitopes, respectively. Successfully sequenced PC and BCP regions showed at least one mutation in 84.6% of the patients. The PC and BCP mutations were G1896A (61%), G1899A (23%), A1762T/G1764A (23%) and G1764T/C1766G (26%). None of the strains with A1762T/G1764A mutation carried the G1764T/C1766G mutant. Conclusions: Our results showed common mutations within HBsAg, occurring in immune epitopes, a high rate of G1896A mutations in the PC region, and a negative correlation between the emergence of A1762T/G1764A mutation and the G1764T/C1766G mutant in the BCP region.