In the present study, we report the isolation of bovine ephemeral fever virus (BEFV) field strains from clinically infected cattle and buffaloes. Twenty Buffy coat samples were collected from each clinically infected cattle and buffaloes in Qaluobia province during the year 2014. The virus was isolated on BHK-21 cell line. Infected cells with BEFV showed 80% cytopathic effect (CPE) after 48 h by the 3rd virus passage. The isolates were serologically identified by neutralization and immunoflourescence techniques, and then molecularly identified using reverse transcription polymerase chain reaction (RT-PCR). Both cattle and buffalo isolates behaved the same pattern for serological and molecular identification in correlation with BEFV Webster reference strain and showed an amplicon size 473 bp for G2 gene. In conclusion, BEFV field strains from cattle and buffaloes seem like but further molecular characterization was required to study their homology.