Abstract Since 2001, alligator farms in the United States have sustained substantial economic losses because of West Nile virus (WNV) outbreaks in American alligators (Alligator mississippiensis). Once an initial infection is introduced into captive alligators, WNV can spread among animals by contaminative transmission. Some outbreaks have been linked to feeding on infected meat or the introduction of infected hatchlings, but the initial source of WNV infection has been uncertain in other outbreaks. We conducted a study to identify species composition and presence of WNV in mosquito populations associated with alligator farms in Louisiana. A second objective of this study was to identify the origin of mosquito blood meals collected at commercial alligator farms. Mosquitoes were collected from 2004 to 2006, using Centers for Disease Control light traps, gravid traps, backpack aspirators, and resting boxes. We collected a total of 58,975 mosquitoes representing 24 species. WNV was detected in 41 pools of females from 11 mosquito species: Anopheles crucians, Anopheles quadrimaculatus, Coquillettidia perturbans, Culex coronator, Culex erraticus, Culex nigripalpus, Culex quinquefasciatus, Mansonia titillans, Aedes sollicitans, Psorophora columbiae, and Uranotaenia lowii. The blood meal origins of 213 field-collected mosquitoes were identified based on cytochrome B sequence identity. Alligator blood was detected in 21 mosquitoes representing six species of mosquitoes, including Cx. quinquefasciatus and Cx. nigripalpus. Our results showed that mosquitoes of species that are known to be competent vectors of WNV fed regularly on captive alligators. Therefore, mosquitoes probably are important in the role of transmission of WNV at alligator farms.