One hundred samples from clinically suspected buffaloes for Bovine Herpes virus -1 infection at Kalubia governorate were subjected for trials of virus isolation on MDBK cell line. Nine positive samples as follows 1 out of 25, 6 out of 25, 1 out of 25 and 1 out of 25 of ocular, nasal, vaginal swabs and tissue samples, respectively. Positive samples upon isolation were identified with indirect fluorescent antibody technique (IFAT) and revealed that 2, 1, and 1 were positive for nasal swabs, vaginal swabs and tissue samples respectively. Polymerase chain reaction (PCR) for glycoprotein B (gB) gene have also been used for molecular identification.