Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex. cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida, and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire E1 protein gene was successfully sequenced from 15 positive pools. Phylogenetic analysis showed that isolates clustered, based on location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, shared amongst all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of Everglades virus in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.