An outbreak of encephalitis due to West Nile (WN) virus occurred in New York City and the surrounding areas during 1999. Mosquitoes were collected as part of a comprehensive surveillance program implemented to monitor the outbreak. More than 32,000 mosquitoes representing 24 species were tested, and 15 WN virus isolates were obtained. Molecular techniques were used to identify the species represented in the WN virus-positive mosquito pools. Most isolates were from pools containing Culex pipiens mosquitoes, but several pools contained two or more Culex species.