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More than 75,000 immature mosquitoes in three genera were collected from coastal California, reared to the adult stage, and tested for virus by plaque assay in Vero cell cultures. Twenty-six strains of Morro Bay (MB) virus, a newly recognized member of the California (CAL) serogroup, were isolated from Aedes squamiger, a pestiferous salt marsh mosquito species restricted to intertidal salt marshes in coastal California and Baja California. The geographic distribution of the isolates was 10 from San Luis Obispo County, one each from Santa Barbara and Orange Counties, and 14 from San Diego County. No virus isolations were made from 23,157 Ae. squamiger collected north of San Luis Obispo County (midpoint in the geographic range of this species in California). Thus, MB virus infection in Ae. squamiger appears to be restricted to the southern range of this species in California. Serum dilution neutralization tests indicated that MB virus represents a novel subtype of the California encephalitis (CE) serotype within the CAL serogroup. Comparative analyses of genomic sequence data from four geographically distinct MB virus isolates indicated that the isolates are genetically similar to each other and distinct from other CE serotype bunyaviruses. Phylogenetic analysis of nucleocapsid protein gene sequence data indicated that MB virus represents a distinct lineage within the CE serotype and thus supports the serologic classification of MB virus as a distinct CAL serogroup virus.