Jamestown Canyon virus (JCV) (family Bunyaviridae, genus Orthobunyavirus) is maintained in a mosquito-deer cycle and has been implicated in the etiology of meningitis and encephalitis with human cases reported from Ontario, Canada, Michigan, Connecticut, and New York. Despite the recognition of symptomatic cases in the northeastern United States, little is known about the genetic relationships of JCV variants circulating in this region. Accordingly, we compared the phylogenetic relationships of 56 JCV isolates from mosquitoes collected in Connecticut over a 40-year period to evaluate their evolutionary history and characterize patterns of genetic diversity in the state. We distinguished at least two major lineages in Connecticut on the basis of phylogenetic reconstruction of small (S), medium (M), and large (L) segment nucleotide sequences. Viruses representing each lineage infected a diverse group of mosquito species over multiple years of sampling and appeared to be geographically structured along an east-west axis. One of these lineages was detected in Connecticut from 1966 through 2006 with few mutational changes accumulating over time. Phylogenetic trees generated from portions of the M and L segments yielded different topologies from S segment sequences as three clades became consolidated into two. Although direct evidence for genetic exchange by reassortment was lacking among cocirculating strains in Connecticut, molecular trees from S, M, and L segments were incongruent, which suggests a distinct evolutionary history or process for each genomic segment. These results suggest that JCV variants are stably maintained in Connecticut where they infect a wide diversity of mosquito species.