Among the avian influenza A virus subtypes, the H5N1 and H9N2 viruses have the potential to cause an influenza pandemic because they are widely prevalent in avian species in Asia and have demonstrated the ability to infect humans. This study was carried out to understand the seroprevalence of H9N2 avian influenza virus in different human population in Boushehr Province which is situated at the South of Iran. Antibodies to H9N2 avian influenza virus in sera from 300 individuals in five different human population in Boushehr province (poultry farm-workers, slaughter-house workers, veterinarians, patients with clinical signs of respiratory disease and normal general citizens were not in contact to poultry at all or only had rare contact) were measured using Hemagglutination-Inhibition (HI) test. The mean antibody titer was found 5.63, 5.1, 4.85, 3.5, 2.73 and seroprevalence was 90, 78.3, 71.6, 46.6 and 25% in these groups, respectively. A higher prevalence was detected in poultry farm-workers, slaughter-house workers and veterinarians possibly enabled by the close and frequent contact of these groups with poultry industry.