Bovine herpesvirus type 1 (BoHV-1), an alphaherpesvirus, is a major pathogen of cattle causing different syndromes such as infectious bovine rhinotracheitis (IBR), infectious pustular vulvovaginitis (IPV) and infectious balanoposthitis (IBP). BoHV-1 control programs have been initiated in several European countries including Germany. One of the major components is the vaccination with inactivated or attenuated glycoprotein E (gE)-deleted live marker vaccines. The aim of this study was the development of a triplex real-time polymerase chain reaction (qPCR) assay for the sensitive, specific and reliable BoHV-1 detection. A BoHV-1-specific glycoprotein D (gD) assay was combined with a gE-specific qPCR system for differentiation between wild-type strains and gE-negative vaccine virus strains. Finally, an internal control based on amplification of the bovine beta-actin gene was introduced to verify efficient DNA extraction and PCR amplification. The analytical sensitivity of the triplex BoHV-1 qPCR enables the detection of 10 genome copies per reaction. Furthermore, the sensitivity of the newly developed qPCR assay was compared to an OIE-validated qPCR and the “gold standard” method of virus isolation in cell culture using 10-fold dilution series of BoHV-1 in extended semen as well as in cell culture medium. For all preparations, the tested qPCR assays showed comparable results and the sensitivity of the triplex qPCR was equal or even greater than that of virus isolation. A broad spectrum of reference strains and field isolates was detected reliably. The specificity of the test was confirmed using nasal swabs, semen and different organ materials of BoHV-1-negative cattle. Bovine herpesviruses type 2, 4 and 5 and further ruminant herpesviruses, namely bubaline herpesvirus (BuHV-1), caprine herpesvirus type 1 and cervine herpesvirus type 1 and 2 (CvHV-1, -2) were tested as well. The close genetic and serological relationship between these viruses combined with their ability to infect bovines may interfere with diagnostics resulting in false-positive results. All non-BoHV-1 herpesviruses were negative in the gD-specific assay, while BuHV-1, CvHV-1 and -2 were tested positive by the gE-specific qPCR. Consequently, the triplex qPCR offers for the first time the possibility to detect some related herpesviruses and distinguish them from BoHV-1 in addition to the simultaneous differentiation of BoHV-1 wild-type and gE-deleted vaccine strains.