The mosquito fauna of Oklahoma has not been evaluated since 1965 and no report has been published concerning species associated with urban areas in the state. Mosquito collections were conducted as part of the West Nile virus (WNV) surveillance program between April and November from 2003 to 2006, using standard collection methods. A total of 74,756 adults were collected in 26 urban centers in 16 counties of Oklahoma. Altogether, 40 species were recorded during this study period, bringing the total mosquito species recorded in Oklahoma to 62 species in 9 different genera and 18 subgenera. An updated checklist of Oklahoma mosquito fauna is included with a comparison to historical records. New state records include 3 species: Aedes muelleri, Anopheles perplexens, and Culex coronator. In addition to updating the checklist, 12 species of mosquitoes were tested for WNV. Pools of Culex pipiens complex represented the highest proportion testing positive for WNV (134/766, 17.5%), followed by Cx. tarsalis (13/192, 6.8%) and Aedes albopictus (5/215, 2.3%). West Nile virus–positive mosquitoes were detected earliest in June 2005 and latest in November 2004. Infected Cx. pipiens complex testing positive for WNV were more prevalent in the eastern and central areas of Oklahoma, whereas positive Cx. tarsalis were found mainly in the western areas of the state. This distinct geographical difference needs to be monitored and followed up to ensure optimal mosquito control efforts in Oklahoma communities with mosquito control capabilities.