Abstract We report the first large-scale serosurvey for West Nile virus (WNV) conducted in the equine population in Iran. Blood samples were obtained in 2008-2009 from 1054 equines collected from 260 districts located in 27 provinces. The overall seroprevalence rate for WNV neutralizing antibodies was 23·7%. Marked geographical variations were observed as province-specific seroprevalence rates ranged from 1% to 88%, the highest values being observed in the southern and western parts of the country. The presence of IgM-positive animals (n=9) indicated a recent circulation of WNV in several provinces. Logistic modelling confirmed this result with a significant effect of age on seropositivity. This study revealed extensive circulation of WNV in Iran particularly in southwestern provinces where the virus probably circulates every year.