The mechanism for long-term maintenance of western equine encephalomyelitis (WEE) virus in California was investigated by studying genetic variation in the E2 portion of the genome of 55 strains of WEE virus isolated since 1938 from different locations in California. Four major lineages were evident: virus strains isolated from the Central Valley since 1993 and Los Angeles in 1991 formed lineage A; southern California strains isolated since 1978 and isolates from the Central Valley from 1978 to 1987 formed lineage B; northern California isolates from 1968 to 1971 formed lineage C; and early isolates from 1938 to 1961 formed a fourth lineage, D. The separation of strains from north and south of the Tehachapi and San Bernardino Mountains (i.e., the Central Valley and southern California, respectively) since 1991 indicates that there has been little recent movement of virus between the two regions and recent strains from these two locations appear to be evolving independently. However, within the Central Valley and within southern California, virus appears to circulate freely, perhaps by movement of birds or mosquito vectors. Although the current virus lineage in the Central Valley may have been introduced from an unknown source in 1991, introduction and establishment of new viral genotypes from outside California do not seem to occur regularly. It appears most likely that virus is maintained in separate geographic areas of California through local persistence in enzootic foci.