Epizootic epitheliotropic disease virus (EEDV) has caused catastrophic losses among hatchery-reared juvenile lake trout Salvelinus namaycush since the early 1980s and remains a major impediment to lake trout restoration in the Great Lakes basin of the USA. Although EEDV has been tentatively designated as a herpesvirus based upon morphological criteria, further characterization of the virus and development of improved detection methods have been hampered by the inability to propagate the virus in cell culture. Recently obtained sequence data for a region of the putative terminase gene from EEDV as well as the related Salmonid herpesvirus 1 and 2 have permitted the development of a polymerase chain reaction (PCR) assay for specific detection of EEDV. The new EEDV PCR demonstrated both an excellent analytic sensitivity and specificity and detected viral DNA as present in the skin of lake trout during periods of active viral outbreaks. In addition, EEDV DNA was detected among healthy appearing juveniles and in the ovarian fluids of spawning adults. Here we describe the development and initial validation steps of the EEDV PCR as a replacement for current diagnostic methods that require virus extraction from the skin, partial purification by isopycnic centrifugation, and visualization of negatively-stained virions by electron microscopy.