A polymerase chain reaction (PCR), utilizing thymidine kinase (tk) primer set of bovine herpesvirus-1, virus isolation (VI) on MDBK cell culture, indirect immunofluorescence assay (IFA) were conducted for detection of BoHV-1 in suspected cattle and buffalo nasal swabs that obtained from four governorates in Egypt (Qalubeya, Menofya, Kafer El-sheikh and Beharia). A total of 9/93 (9.6%) of cattle and 4/65 (6.2%) of buffalo nasal swabs were BoHV-1 positive in this study. PCR was superior to immunodetection using IFA after VI procedure in cell culture. Besides, it was the most discriminative assay that detected BoHV-1 viral DNA extracted directly from (100%) of buffalo and (88.9) of cattle clinical specimens. These PCR negative viral isolates were regarded as antigenically related, yet genetically distinct, other ruminant herpesviruses that require further studies. Findings of this study emphasized the BoHV-1 tk based PCR as a sensitive, discriminative and rapid tool for detection of BoHV-1 infections, without confusion with other ruminant herpesviruses.