Bovine ephemeral fever (BEF) is an arthropode borne rhabdovirus, which causes a disabling febrile infection of cattle and water buffalo. A sever outbreak was recorded in Egypt during 2000 and 2001. Using RT PCR, a primer designed to amplify 438 nucleotide fragments from the glycoprotein G gene allowed specific amplification of BEFV-cell culture isolates from Egypt (Menoufia and Alexandria Governorates) and Japan. The PCR product was sequenced and analyzed. PCR product of Alexandria isolate was cloned and labeled with digoxigenin and used as diagnostic probe for BEF virus infection using dot-blot hybridization. Thirty six samples were collected from Menoufia Governorate (Berket El-Sabaa, Tokh Tambesha and Salamon regions) and Demiate Governorate and were tested in dot-blot hybridization. BEFV was detected in 24 out of 36 samples (66.7%) as follow: 9/9 (100%) correspond to Berket El-Sabaa and Tokh Tambesha regions and 3/9 (33.3%) correspond to Demiate and Salamon regions. In conclusion, a local Egyptian probe was designed and applied in the dot blot hybridization technique for diagnosis of bovine ephemeral fever virus.