A polymerase chain reaction (PCR) based on a combination of oligonucleotide primers selected using the octamer frequency disparity method with primers specific for EHV-5 (described by other authors) recognized all of a series of gamma herpesvirus field isolates. This PCR produced only three fragments: (1) one EHV-2-specific; (2) one EHV-5-specific; and (3) a fragment that occurred alone or in combination with the other two. Cloning and sequencing of four different isolates yielding only the last PCR product showed that this corresponds to a deletion/insertion mutant of EHV-2. The fact that this mutant was also plaque-purified from a culture producing all three PCR fragments demonstrated that the virus producing this fragment was distinct from the other two and that this specific DNA fragment was not an artefact due to PCR amplification. These data show that equine gamma herpesviruses are genetically more heterogeneous than previously assumed. The PCR failed to directly detect gamma herpesviruses from the DNA extracted from the same starting material used for the isolation of gamma herpesvirus by cocultivation with indicator cells. This demonstrates that the most reliable method for detection of equine gamma herpesviruses is the cocultivation with indicator cells.