Objective: To evaluate the prevalence of West Nile virus seropositivity in the general population of Mashhad, Northeast of Iran. Methods: One hundred and eighty two individuals living in the city of Mashhad were studied using cluster sampling method. Both IgM and IgG antibodies against WNV were detected by ELISA method. Results: In this study, the overall IgG seroprevalence of positive West Nile virus was 11%; however, IgM antibody was not found in the participants. Conclusions: Our study suggested that the prevalence rate of West virus is considerable in Mashhad city. It seems necessary for clinicians and health care workers to be aware of WNV infection in the Northeast Iran.