Epizootic hemorrhagic disease virus (EHDV) causes a highly infectious noncontagious hemorrhagic disease in wild and captive deer (Cervidae) populations in the US. Although rapid and accurate identification of the disease is important, identification of the serotype is equally important for understanding the epidemiology of the disease in white-tailed deer (Odocoileus virginianus) populations. We developed a one-step multiplex reverse transcriptase PCR assay for rapid differentiation and identification of EHDV serotypes 1, 2, and 6 in cell culture and clinical samples by targeting the viral gene segment 2 (L2) that encodes for the structural protein VP2. From 2009 to 2012, 427 clinical samples including tissue and blood (in ethylenediaminetetraacetic acid) from white-tailed deer, found EHDV positive by real-time PCR, were used to evaluate this subtyping assay. Eighteen percent of the positive samples tested were EHDV-1, 59% were EHDV-2, and 21% were EHDV-6; 2% of the samples were positive for more than one subtype, indicating mixed infection. This assay provides a rapid, sensitive, specific diagnostic tool for differentiation and identification of EHDV serotypes in field samples and virus isolates.