We previously demonstrated that a Venezuelan equine encephalitis virus (VEEV) DNA vaccine that was optimized for increased antigen expression and delivered by intramuscular (IM) electroporation (EP) elicits robust and durable virus-specific antibody responses in multiple animal species and provides complete protection against VEEV aerosol challenge in mice and nonhuman primates. Because our ultimate goal is to develop a single multi-agent vaccine formulation that can elicit protection against VEEV, western equine encephalitis virus (WEEV), and eastern equine encephalitis virus (EEEV), here we performed a comparative evaluation of the immunogenicity and protective efficacy of individual optimized VEEV, WEEV, and EEEV DNA vaccines with that of a 1:1:1 mixture of these vaccines, which we have termed the 3-EEV DNA vaccine, when delivered by IM EP. The individual DNA vaccines and the 3-EEV DNA vaccine elicited robust and durable virus-specific antibody responses in mice and rabbits and completely protected mice from homologous VEEV, WEEV, and EEEV aerosol challenges. In addition, these DNA vaccines provided protection in mice that was similar to that of the respective live-attenuated VEEV vaccine and superior to that of the respective formalininactivated WEEV and EEEV vaccines currently used in humans under Investigational New Drug status. Taken together, the results from these studies demonstrate that the individual VEEV, WEEV, and EEEV DNA vaccines and the 3-EEV DNA vaccine delivered by IM EP provide an effective means of eliciting protection against lethal encephalitic alphavirus infections in a murine model and represent viable next-generation vaccine candidates that warrant further development.