Background: One of the challenges to treat hepatitis C virus (HCV) infection is the activation of hepatitis B virus (HBV) that occurs during treatment of HCV infection with direct-acting antivirals (DAAs) in some patients. The detection of serum or liver HBV DNA in the absence of serum HBsAg (HBV surface antigen) is described as occult HBV infection (OBI). Objectives: The current study aimed at determining the prevalence of OBI in Iranian DAA-naïve HCV-infected patients with hemophilia. Methods: The current study was conducted on 100 patients with hemophilia receiving DAAs. The sera obtained from these patients were tested for the presence of HBsAg. Then, the presence of the HBV DNA was detected in peripheral blood mononuclear cell (PBMC) and also plasma samples using nested polymerase chain reaction (PCR). Results: Among the 100 study subjects, 81 (81%) were male and 19 (19%) female. The mean age of the patients was 37 ± 10.50 years. All patients had previously received HBV vaccine. In the current study, HBV DNA was observed in 1% of plasma and in 3% of PBMC samples. In addition, none of the patients who had positive result for HBV detection test previously had markers of HBV infection (anti-HBc (HB core antigen) antibodies, anti-HBs antigens, and positive result of DNA PCR) and all had negative results for HCV RNA after treatment. Conclusions: Generally, the prevalence of OBI was low, but however, HBsAg negativity was not sufficient to completely exclude the presence of HBV DNA. Thus, the serological markers of HBV infection should be confirmed by molecular tests for the presence of possible occult infection.