Bovine viral diarrhea virus (BVDV) infections are enzootic in the cattle population and continue to cause significant economic losses to the beef and dairy industries worldwide. Extent of the damages has stimulated increasing interest in control programs directed at eradicating BVDV infections. Use of a BVDV marker vaccine would facilitate eradication efforts as a negatively marked vaccine would enable differentiation of infected from vaccinated animals (DIVA). We describe here the construction of three chimeric BVDVs containing glycoprotein E(rns) of heterologous pestiviruses and the evaluation of the chimera viruses as potential marker vaccines against BVDV infections. Chimeric NADL/G-E(rns), NADL/R-E(rns), and NADL/P-E(rns) were constructed by replacing the E(rns) gene of the full-length BVDV (NADL strain) genome with the E(rns) genes of giraffe (G-E(rns)), reindeer (R-E(rns)), or pronghorn antelope (P-E(rns)) pestiviruses, respectively. Each chimeric NADL virus was viable and infectious in RD 420 (bovine testicular) and BK-6 (bovine kidney) cells. By immunohistochemistry assays, NADL/G-E(rns) and NADL/R-E(rns) chimeric viruses reacted to BVDV E(rns) specific monoclonal antibody (mAb) 15C5, whereas the NADL/P-E(rns) chimeric virus did not. In an animal vaccination study, inactivated vaccines made from two chimeric viruses and the wild type NADL BVDV induced similar neutralizing antibody responses. NADL/P-E(rns)-vaccinated animals were distinguished from animals vaccinated with the wild type virus by means of a companion serological DIVA assay. These results show that chimeric NADL/P-E(rns) virus containing the E(rns) gene of pronghorn antelope pestivirus could be a potential marker vaccine candidate for use in a BVDV control and eradication program.