Crows have been the centerpiece of avian West Nile virus (WNV) surveillance and research in North America. This work has demonstrated variation in susceptibility to WNV infection between American (Cor vus brachyrhynchos) andFish Crows (Corvus ossifragus). The higher WNV-associated mortality rate in American Crows compared with Fish Crows suggests that WNV antibody prevalence would be greater in the Fish Crow population. The objectives of this study were to 1) determine whether Fish Crows had higher WNV antibody prevalencethan American Crows, 2 ) determine th e persistence o f antibodies to WNV in naturally infected Fish Crows, and 3) develop a technique to distinguish Fish Crows from American Crows on the basis of sequence analysis and restriction enzyme digestion of a mitochondrial DNA fragment. West Nile virus antibody prevalence was 16.5% (n = 97) in Fish Crows and 5.7% in American Crows (n = 53) collected from Georgia between 2004 and 2006. Antibodies persisted at high titers for 12 mo in Fish Crows. This is the first report of WNV antibody persistence in a crow species. A polymerase chain reaction technique paired with restriction enzyme digestion easily distinguished American Crows from Fish Crows on the basis of a mitochondrial DNA fragment.