Hepatitis B virus vaccination was recommended for HIV patients. Despite the relative success of hepatitis B virus vaccination, breakthrough infections can occur infrequently in patients, and it can be due to occult hepatitis B virus infection, vaccine unresponsiveness and/or emergence of escape mutants. This study assessed the presence of occult hepatitis B virus infection and S gene escape mutants in HIV-positive patients after hepatitis B virus vaccination. Ninety-two human immunodeficiency virus patients were enrolled in this study including 52 responder to hepatitis B virus vaccine and 40 non-responders. All of the cases received hepatitis B virus vaccine according to routine hepatitis B virus vaccination protocol. The presence of hepatitis B virus-DNA was determined by real-time polymerase chain reaction. In hepatitis B virus-DNA positive samples, the most conserved regions of S gene sequences were amplified by nested polymerase chain reaction and polymerase chain reaction products were sequenced. Occult hepatitis B virus infection was detected in two cases. Glycine to arginine mutation at residue 145 (G145R) within the ‘a’ region of the S gene was detected in one of the occult hepatitis B virus infection cases who was in non-responder group. This study showed that the prevalence of occult hepatitis B virus infection and vaccine escape mutants was low in our hepatitis B virus-vaccinated human immunodeficiency virus patients in both responder and non-responder groups. So there was no alarming evidence indicating breakthrough hepatitis B virus infection in our vaccinated human immunodeficiency virus cases.