ABSTRACT This study was performed to determine the prevalence, genotype distribution and risk factors of hepatitis B virus (HBV) infection among β-thalassemia patients. ELISA was used to detect HBsAg and HBcAb. Molecular evaluation of HBV infection was performed by nested PCR, targeting S, X and pre-C regions of the genome, and sequencing. Of 126 thalassemia patients, 4 cases (3.17%) were positive for HBsAg, 23 cases (18.25%) were positive for HBcAb, and 6 cases (4.76%) had HBV viremia with genotype D, sub-genotype D3 and subtype ayw2. HBV prevalence among thalassemia patients was not statistically associated with gender distribution, place of residency, marital status and frequency of blood transfusion. HBsAg seroprevalence was significantly higher in Afghan immigrants and patients with ALT levels of 41–80 IU/L. The prevalence of HBV viremia was significantly higher among thalassemia patients aged >20 years compared to the patients aged <20 years. Moreover, 1.59% of thalassemia patients had seropositive occult HBV infection, which was positive for HBV-DNA and HBcAb but negative for HBsAg. Considering the relatively high prevalence of occult HBV infection among thalassemia patients, there is a possibility of their contamination through donated blood. Therefore, screening of donated blood based on detection of HBsAg cannot abolish HBV transmission through blood transfusion.