Eastern equine encephalitis virus (EEEV), the sole species in the EEE antigenic complex, is divided into North and South American antigenic varieties based on hemagglutination inhibition tests. Here we describe serologic and phylogenetic analyses of representatives of these varieties, spanning the entire temporal and geographic range available. Nucleotide sequencing and phylogenetic analyses revealed additional genetic diversity within the South American variety; 3 major South/Central American lineages were identified including one represented by a single isolate from eastern Brazil, and 2 lineages with more widespread distributions in Central and South America. All North American isolates comprised a single, highly conserved lineage with strains grouped by the time of isolation and to some extent by location. An EEEV strain isolated during a 1996 equine outbreak in Tamaulipas State, Mexico was closely related to recent Texas isolates, suggesting southward EEEV transportation beyond the presumed enzootic range. Plaque reduction neutralization tests with representatives from the 4 major lineages indicated that each represents a distinct antigenic subtype. A taxonomic revision of the EEE complex is proposed.