Mosquitoes collected during 2003, 2004, and 2005 in Alberta, Canada, were screened for the presence of a wide range of arboviruses by reverse transcription-polymerase chain reaction (RT-PCR). Nucleic acid extracts from mosquito slurries were amplified using universal primers designed to detect viruses belonging to the Flavivirus genus of the Flaviviridae family and California and Bunyamwera serogroups of the Bunyavirus genus within the Bunyaviridae family. Species-specific detection of Western equine encephalitis virus and Eastern equine encephalitis virus was also performed. Amplified products were analyzed, and the viral target was identified by sequencing. Of the 418 pools tested, 3 pools contained Cache Valley virus belonging to Bunyaviridae and 103 pools were positive for a previously undescribed flaviviral sequence that was most similar to Kamiti River virus. These data suggest that nucleic acid amplification using broadly reactive primers can be adopted for arbovirus surveillance in mosquito populations, and this approach has the potential to detect both previously recognized and novel viruses.