Recurring infection of reticuloendotheliosis virus (REV), an avian oncogenic gammaretrovirus, has been a major obstacle in attempts to breed and release the endangered Attwater's prairie chicken (Tympanicus cupido attwateri). The aim of this study was to develop a DNA vaccine that protects the birds against REV infection. A plasmid was constructed expressing fusion proteins of REV envelope (env) and VP22 of Gallid herpesvirus 2 or REV gag and VP22. Birds vaccinated with these recombinant plasmids developed neutralizing antibodies; showed delayed replication of virus; and had significantly less infection of lymphocytes, specifically CD4 lymphocytes. Although the vaccine did not prevent infection, it offered partial protection. Birds in field conditions and breeding facilities could potentially benefit from increased immunity when vaccinated.