Epizootic subtype IAB and IC Venezuelan equine encephalitis viruses (VEEV) readily infect the epizootic mosquito vector Aedes taeniorhynchus. The inability of enzootic subtype IE viruses to infect this mosquito species provides a model system for the identification of natural viral determinants of vector infectivity. To map mosquito infection determinants, reciprocal chimeric viruses generated from epizootic subtype IAB and enzootic IE VEEV were tested for mosquito infectivity. Chimeras containing the IAB epizootic structural gene region and, more specifically, the IAB PE2 envelope glycoprotein E2 precursor gene demonstrated an efficient infection phenotype. Introduction of the PE2 gene from an enzootic subtype ID virus into an epizootic IAB or IC genetic backbone resulted in lower infection rates than those of the epizootic parent. The finding that the E2 envelope glycoprotein, the site of epitopes that define the enzootic and epizootic subtypes, also encodes mosquito infection determinants suggests that selection for efficient infection of epizootic mosquito vectors may mediate VEE emergence.