BackgroundImmunomodulators and Nucleotide analogues have been used globally for the dealing of chronic hepatitis B virus (HBV) infection. However, the development of drug resistance is a major limitation to their long-term effectiveness.ObjectivesThe aim of this study was to characterize the hepatitis B virus reverse transcriptase (RT) protein variations among Iranian chronic HBV carriers who did not receive any antiviral treatments.Materials and MethodsHepatitis B virus partial RT genes from 325 chronic in active carrier patients were amplified and directly sequenced. Nucleotide/amino acid substitutions were identified compared to the sequences obtained from the database.ResultsAll strains belonging to genotype D.365 amino-acid substitutions were found. Mutations related to lamivudine, adefovir, telbivudine, and entecavir occurred in (YMDD) 4% (n = 13), (SVQ) 17.23% (n = 56), (M204I/V + L180M) 2.45% (n = 8) and (M204I) 2.76% (n = 9) of patients, respectively.ConclusionsRT mutants do occur naturally and could be found in HBV carriers who have never received antiviral therapy. However, mutations related to drug resistance in Iranian treatment-naïve chronic HBV patients were found to be higher than other studies published formerly. Chronic HBV patients should be monitored closely prior the commencement of therapy to achieve the best regimen option.