In a previous study, a new flavivirus genome sequence was identified in Culex tarsalis mosquitoes obtained in Alberta, Canada and was shown to be genetically related to but distinct from members of the insect-specific flaviviruses. Nonstructural protein 5-encoding sequences amplified from Cx. tarsalis pools from western Canada have shown a high similarity to genome sequences of novel flaviviruses isolated from mosquitoes in California and Colorado. Despite wide distribution of this virus, designated Calbertado virus, strains demonstrate a high degree of nonstructural protein 5 nucleotide (> 90%) and amino acid (> 97%) identity. The ecology and geographic range of Calbertado virus warrants further study because it may potentially influence transmission of mosquito-borne flaviviruses, including important human pathogens such as West Nile and Saint Louis encephalitis viruses.