Outbreaks of West Nile virus on a Florida alligator farm prompted an investigation of which species of mosquitoes were feeding on the animals at the farm. Mosquitoes were collected on 4 separate overnight trips in September and October 2003 by using CO2-baited Centers for Disease Control light traps and wooden resting boxes that were placed inside or near the alligator housing pens. Mosquitoes were identified to species, bloodfed individuals were separated, and their abdomens were removed for DNA extraction. The DNA was tested to determine the vertebrate origin of the blood meal by polymerase chain reaction (PCR) amplification by using 4 primer sets specific to crocodilians, alligators, mammals, and birds. PCR products were sequenced to identify hosts. Of the 37 mosquito blood meals tested, 13 blood meals were positively identified to species, and 7 blood meals of those 13 were from Alligator mississippiensis, the American alligator. Alligator blood was found in Culex erraticus, Mansonia dyari, and Ma. titillans, and to our knowledge, this represents the first report of these mosquito species feeding on American alligators.