Bovine ephemeral fever virus (BEFV) is an economically important vector-borne pathogen of cattle in tropical and sub-tropical regions of Australia, Asia, Africa and the Middle East. Although clinical cases of bovine ephemeral fever are usually attributed to BEFV, definitive diagnosis is rarely performed and at least two other related viruses, kotonkon virus (KOTV; an ephemerovirus) and Fukuoka virus (FUKAV; an unassigned rhabdovirus), can cause similar clinical signs. As vaccines have been developed against BEFV but not against KOTV or FUKAV, a test capable of detecting and differentiating these pathogens would be useful. In the present study, an RT-PCR method using degenerate primers designed to a region of block III of the polymerase (L) gene was developed and optimised for primer annealing temperature and MgCl2 concentration. The RT-PCR detected all known ephemeroviruses and several other closely related insect-transmitted rhabdoviruses, including FUKAV. Viruses could be identified by subsequent sequencing and genetic analysis of the amplicons. BEFV could be detected using both tissue culture isolates and cattle blood to a sensitivity of 500 RNA copies per reaction. This test will be useful for establishing the identity of the causative agent of bovine ephemeral fever from field samples and cultured isolates.