West Nile Virus (WNV) causes significant mortalities in captive-reared American alligators (Alligator mississippiensis). Alligators can amplify the virus, serve as a reservoir host, and represent a source of infection for humans. A killed WNV vaccine is commercially available for use in alligators. In 2014, a case of suspected WNV infection from an alligator farm in Louisiana revealed that tissues of previously vaccinated animals were positive for WNV by reverse transcriptase polymerase chain reaction (RT-PCR), whereas nonvaccinated animals were negative. Further testing demonstrated that the RT-PCR could detect viral RNA in the vaccine product itself. Virus isolation was negative in the tissues and the vaccine. These findings suggest that the RT-PCR detects the viral RNA from the killed vaccine in the tissues.