Bovine viral diarrhea virus (BVDV) is the most prevalent infectious disease of cattle. It causes financial losses from a variety of clinical manifestations and is the subject of a number of mitigation and eradication schemes around the world. This study was designed for isolation and identification of BVDV in Kalubeya governorate. The study was carried out on 400 Buffy coat and tissue samples from cattle and buffaloes. Direct detection of BVDV antigen by antigen capture enzyme linked immunosorbent assay (AC-ELISA) showed positive results in 47.4% (95) and 10.5% (21) in cattle and 38% (76) and 6% (12) in buffaloes for buffy coat and tissue samples, respectively. Virus isolation (VI) on MDBK cell culture revealed negative results which subjected to indirect fluorescent antibody technique (FAT), revealed characteristic intracytoplasmic apple green fluorescence indicating presence of non-cytopathogenic strain of BVDV. Molecular detection using reverse transcription-polymerase chain reaction (RT-PCR) revealed the presence of specific PCR product at the correct expected size of the BVDV genotype I (190 bp)