Sera of 624 blood donors were evaluated to determine seroprevalence of West Nile virus (WNV) antibodies following the 2003 WNV epidemic in Nebraska. Geographic factors contributing to differences in WNV seropositivity were evaluated. The overall prevalence of WNV in Nebraska was higher than reported previously in other U.S. locations (9.5% WNV immunoglobulin G seroprevalence rate), with the highest prevalence identified in the western part of the state (19.7%), followed by the central (13.8%) and the eastern (4.2%) parts. Regions of the state with the highest WNV-positive mosquito rates correlated with the highest human WNV seroprevalence rates. The results showed that both the western and central parts of the state, where mosquito positivity rates were highest, had significantly higher seroprevalence rates than the eastern region. Additional studies are needed to determine whether the high prevalence rates in Nebraska will be reflected in other states and what impact environmental and geographical factors may have on future outbreaks of WNV infection.