This study was carried out to determine the seroprevalence of hepatitis D virus among HBsAg positive individuals in the northeast part of Iran. One hundred thirty nine HBsAg positive subjects detected from a population based single stage cluster sampling in Golestan province of Iran were enrolled. All cases were evaluated for the presence of anti-HDV antibodies using commercially available ELISA kits. Logistic regression was used to determine the relationship between independent variables and HDV seropositivity. Of 139 cases, 68 (48.9%) were males and 71 (51.1%) were females. The mean age was 41.89 +/- 11.30 years (25-64 years). Anti-HDV antibody was positive in 8 (5.8%) subjects with female predominance (9.9% versus 1.5%, p = 0.06; odds ratio = 7.32, 95% CI: 0.87-61.23). No significant relationship was seen between anti-HDV seropositivity and demographic factors such as age, place of residence and marital status. These findings showed that HDV infection was endemic in Golestan province (northeast) of Iran. Seroprevalence of Anti-HDV in the present study was higher than some previous studies from other parts of Iran. Our results suggest that the prevalence of HBV/HDV co-infection in Iran has increased during the last decade. Therefore, practitioners and all health care managers should be made aware of the risk of dual infection with HBV and HDV.