Technical Abstract: Malignant catarrhal fever (MCF), an often fatal disease primarily of ruminant species, is caused by a group of herpesviruses in the Gammaherpesvirinae subfamily. The absence of a test for simultaneous detection and differentiation of MCF viruses presents a difficulty in the diagnosis of MCF especially in those cases from mixed species operations. In this study, we developed a single-tube multiplex real-time PCR using one pair of conserved primers, which amplifies a region of the viral DNA polymerase gene, in conjunction with fluorescent probes specific for each unique sequence of five known pathogenic MCF viruses: ovine herpesvirus 2, alcelaphine herpesvirus 1, caprine herpesvirus 2, white-tailed deer-MCF virus, and the MCF virus in ibex. The assay was able to detect as little as 50 copies of each virus target and we observed no cross reactivity among the probes or with other common ruminant herpesviruses. High sensitivity (98.2%) and specificity (100%) were obtained by testing a panel of samples known to be either positive (n=57) or negative (n=34) to the viruses of interest. The multiplex real-time PCR reported in this study is rapid and reliable for simultaneously detecting and differentiating MCF viruses in samples from clinically affected animals, which is of great significance to diagnosing and controlling MCF. Moreover, the assay was developed in a flexible platform, which can be adjusted according to the interests of different laboratories. The technology described here can also potentially be applied to differential diagnosis of other closely related viruses especially herpesviruses in human and animals.