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Theileria orientalisIkeda is a newly identified agent of bovine infectious anemia in the United States. Although T. orientalisIkeda is transmitted by ticks other than the tick that transmits Anaplasma marginale—a bacterial etiology of bovine infectious anemia—the geographic distributions of these 2 infectious organisms overlap, with coinfection reported in some cattle. Only anaplasmosis has an approved effective treatment in the United States. To provide rapid diagnostic information for producers with anemic animals, we developed a duplex real-time PCR (rtPCR) for A. marginaleand T. orientalis. With a cutoff of 38 cycles, the duplex assay has a sensitivity of 97.0% and a specificity of 100% for A. marginale; with a cutoff of 45 cycles, the duplex assay has a sensitivity and a specificity of 100% for T. orientalis, compared to existing tests. In addition to providing a tool for improved clinical decision-making for veterinarians and producers, our rtPCR facilitates the study of coinfection of cattle in Virginia. Of 1,359 blood samples analyzed, 174 were positive for T. orientalis, 125 were positive for A. marginale, and 12 samples were positive for both T. orientalisand A. marginale.Hence, coinfection by these 2 agents of bovine infectious anemia does occur within Virginia. It is likely that this pattern of infection will be seen in other regions where T. orientalisand A. marginaleinfections are endemic, despite the difference in tick vectors.