The aim of this study is to investigate the presence of Bovine Ephemeral Fever (BEF) virus in EDTA-treated blood, sera, and spleen samples of 228 suspected animals originated from 5 different provinces of Turkey in 2020, as well as to perform molecular characterization and phylogenetic analysis of positive cases. For this purpose, 79 whole blood and 2 spleen samples as well as 168 sera samples from cattle varying in breed, age, and sex,were examined by using Real Time RT-PCR and Blocking ELISA, respectively. Two new degenerate primers amplifying 956 bp of the protein G (AVKEF_AATGTTCCNGTGAATTGTGGAG and AVKER_TGCATAATCYCTTCCTGGTCT) were designed for RT-PCR testing and the phylogenetic analysis of positive samples was performed. 64.20% (52/81) of the defibrinated blood (63.29%, 50/79) and spleen (100%, 2/2) samples, and 69.04% (116/168) of sera samples resulted positive by RT-PCR. By using VERO cell culture, BEF virus was isolated from blood (n=1) and spleen(n=2) tissues in Adana and Şanliurfa provinces. The phylogenetic analysis revealed that the BEF virus circulated in Turkey during the 2020 regional epidemic belongs to the Middle East lineage, which has its significance for the selection of proper vaccine and the control of the disease.