Technical Abstract: Bluetongue virus (BTV) causes disease in domestic and wild ruminants resulting in significant economic loss. The closely related Epizootic hemorrhagic diseases virus (EHDV) has been associated with bluetongue-like disease in cattle. Although US EHDV strains have not been experimentally proven to cause disease in cattle there is serologic evidence of infection in cattle. Therefore rapid diagnosis and differentiation of BTV and EHDV is required. The genetic sequence information and bioinformatic analysis necessary to design a real-time reverse transcriptase polymerase chain reaction assay for the early detection of indigenous and exotic BTV and EHDV is described. This foundation was accomplished by phylogenetic analysis of two conserved target genes; one that is highly expressed in infected mammalian cells, the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design was necessary for a comprehensive gene amplification diagnostic test. This information has been used as the basis for the development of rapid multiplex BTV/EHDV real-time PCR that detects all known serotypes and distinguishes between BTV and EHDV. The sensitivity of the assay is sufficient for a rapid single tube diagnostic application without the contamination problems associated with standard PCR and especially nested-PCR tests.