Urbanization is a widespread phenomenon that is likely to influence the prevalence and impact of wildlife pathogens, with implications for wildlife management and public health policies toward zoonotic pathogens. In this study, wild songbird populations were sampled at 14 sites along an urban rural gradient in the greater metropolitan Atlanta (Georgia, USA) area and tested for antibodies to West Nile virus (WNV). The level of urbanization among sites was quantitatively assessed using a principal component analysis of key land use characteristics. In total, 499 individual birds were tested during the spring and summer over three years (2004-2006). Antibody prevalence of WNV increased from rural to urban sites, and this trend was stronger among adult birds relative to juveniles. Furthermore, antibody prevalence among Northern Cardinals (Cardinalis cardinalis) was significantly higher than in other songbird species along the urban gradient. Findings reported here indicate that ecological factors associated with urbanization can influence infection patterns of this vector-borne viral disease, with likely mechanisms including changes in host species diversity and the tolerance or recovery of infected animals.