In 2002, more than 17,000 mosquito pools collected in Canada (Quebec, Ontario, and Manitoba) were tested at the National Microbiology Laboratory in Winnipeg, Manitoba, for infection with West Nile virus (WNV). Using real-time reverse-transcriptase polymerase chain reaction (RT-PCR), 558 mosquito pools (86% Culex species and 14% other species) had evidence of infection with WNV. Only 30% of the Culex specimens, however, were identified to the species level. In this study, Culex species-specific PCR primers were designed to identify individual mosquitoes and mixed pools of Culex mosquitoes to species. In addition, pools of non-Culex mosquitoes that tested positive for WNV were also screened for Culex DNA to determine the frequency of cross-contamination among mosquitoes of different species. All DNA extracts from 121 Culex and 51 non-Culex pools, previously positive for WNV, were screened, and Culex DNA was detected in approximately 6% of non-Culex pools. This study demonstrates that contamination among mosquito species can occur and emphasizes that precautions should be taken to minimize this potentially confounding effect.