Epizootic strains of Venezuelan equine encephalitis virus (VEEV) cause epidemics by exploiting equines as highly efficient amplification hosts for mosquito transmission. Although phylogenetic studies indicate that epizootic VEEV strains emerge via mutation from enzootic progenitors that are incapable of efficient equine amplification, the molecular mechanism(s) involved remain enigmatic. The convergent evolution of E2 envelope glycoprotein mutations suggests that they are critical to VEEV emergence, but little is known about the role of non-envelope genes. We used the guinea pig, the small animal model that best predicts the ability to generate equine viremia, to assess the role of envelope versus other mutations in the epizootic phenotype. Using reciprocal chimeric viruses generated by swapping the envelope genes of closely related epizootic IC and enzootic ID strains, infections of guinea pigs demonstrated that envelope and non-envelope genes and sequences both contributed to virulence. However, early replication in lymphoid tissues appeared to be primarily envelope dependent. Copyright © 2005 by The American Society of Tropical Medicine and Hygiene.