BACKGROUND Prevalence of hepatitis B virus (HBV) infection has been reported to be higher in the institutionalized mentally disabled patients than that of the general population previously reported in Iran. This study aims to investigate HBV infection among nurses and families of the hospitalized mentally disabled patients. METHODS This study was conducted on 110 nurses and family members of the mentally disabled patients who were hospitalized in five residential care centers of Tehran. The presence of hepatitis B surface antigen (HBsAg) and hepatitis B core antibody (HBcAb) was examined using the enzyme-linked immunosorbent assay (ELISA). Afterwards, HBV DNA was extracted, and then propagated via a nested polymerase chain reaction (PCR) and specific primers. Finally, a phylogenetic tree was constructed using the neighbor-joining method to compare virus genomes in the nurses' serum with other isolated HBVs worldwide. RESULTS Out of 102 studied nurses, three (3%) were positive for HBsAg (100% female). Also, no patient was positive for the HBV genome, while eight (7.3%) nurses were positive for HBcAb including two (25%) males and six (75%) females. Genome sequencing of one DNA positive sample showed that the isolated virus from this patient contained sub genotype D1 and subtype ayw2. The results of none of the family members were positive for HBsAg, HBcAb, or HBV DNA. CONCLUSIONS This study showed a higher prevalence of HBsAg among nurses (3%) compared to the Iranian general population (1.7-2.1%). The virus isolated from the nurses belonged to subgenotype D1 and subtype ayw2 in accordance with previous Iranian reports. Also, there was no drug-resistant or vaccine-escape mutations in the obtained viral genome. Moreover, low immune pressure on the virus in the asymptomatic chronic HBV patients might be responsible for low nucleotide divergence among the derived HBV genome.