In the present study, a total of 600 cattle and buffalo were selected from three different topographical locations of Punjab on the basis of clinical biomarkers. Blood and mosquito samples were collected. Blood samples were processed through complement fixation test and RT-PCR, while mosquito samples were processed through RT-PCR. Overall prevalence of bovine ephemeral fever (BEF) in indigenous breeds of bovine was 63.33%, lower, than exotic breeds 64.66% which were found to be more prone to disease. In the case of buffalo breeds, prevalence was more in Nili Ravi buffalo, i.e. 36.6% than Kundi breed, i.e. 33.3%. Prevalence of disease was found maximum in humid and plain area of Lahore, i.e. 56%. In dry, dusty and hot weather of Multan overall the prevalence of disease was 55.5%, while in Rajanpur topography of the land is mixed comprising of riverine, plateau, mountainous and semi-hilly areas showed 52.2% prevalence. When immunity status was taken into account it was found that vaccinated animals showed very low prevalence, i.e. 4.39% as compared to non-vaccinated animals having 66.53%. Another risk factor age suggested that younger animals were more affected (60.17%) as compared to older ones (29.41%). It was also observed that animals in closed housing system were more affected as compared to open housed animals. When comparative efficacy of diagnostic tests was done then the results of RT-PCR was found significant (P>5), than complement fixation test.