Background: Hepatitis B virus (HBV) and hepatitis D virus (HDV) infections are known as major health concerns worldwide. Nucleoside analogs (NAs) are widely used in anti-HBV therapy. However, HBV resistance to Nas may result in disease recurrence. Objectives: The present study aimed to evaluate the prevalence of HBV and HDV infections, and to investigate the presence of YMDD motif in HBV-infected patients. Methods: In this cross-sectional study, a total of 1600 blood specimens were collected from HBV-suspected patients, who referred to diagnostic centers in Qom Province, Iran from July 2018 to March 2019 before and after lamivudine therapy. The presence of HBV in the specimens was investigated using ELISA and nested PCR assays. Also, HDV and YMDD motif were detected in HBV-infected patients using the PCR technique. In order to confirm the presence of HDAg gene of HDV, the PCR products of some HBV/HDV-positive specimens were subjected to direct sequencing. Results: The number of HBsAg positive patients was 180 (11.25%) (P < 0.05) according to ELISA and nested PCR assays; these patients were in the age range of 32-38 years. Also, 11 (6.1%) patients were infected with HBV/HDV. The frequency of lamivudine resistance mutation in the YMDD region was 28 (15.5%) among chronic HBV patients. The constructed phylogenetic trees for HDAg gene of HDV showed that most HBV/HDV isolates had similar sequences to the Iranian isolates. Conclusions: The present results showed that it is important to increase our knowledge about the frequency of chronic HBV and HDV infections. Also, development of new antiviral medications is important for prevention and prompt and appropriate treatment.