•Multiple diagnostic kits confirm CCHF prevalence in Ugandan cattle.•CCHF is circulating in areas with no history of human cases in Uganda.•Human CCHF cases in Uganda do not well overlap with the presence of Hyalomma ticks.•Investigation of the true incidence of human CCHF cases in Uganda is required. Crimean-Congo Hemorrhagic Fever (CCHF) is a severe tick-borne viral hemorrhagic disease caused by Crimean-Congo Hemorrhagic Fever Virus (CCHFV) that poses serious public health challenges in many parts of Africa, Europe and Asia. We examined 500 cattle sera samples from five districts for CCHFV antibodies using in-house and commercially available (IDVet) ELISA, Immunofluorescent assay (IFA) and Real-time polymerase chain reaction (RT-PCR). 500 cattle (73.8 % females) were analyzed; CCHFV seropositivity was 12.6 % (n = 63) and 75.0 % (n = 375) with the in-house and IDVet ELISAs, respectively. Seropositivity was associated with geographical location, increasing age, being female, and having a higher tick burden. Twenty four out of the 37 (64.8 %) were seropositive for CCHFV using IFA and all were negative for virus on RT-PCR. The IFA results were more comparable to IDVet (κcoefficient = 0.88, p = <0.01) than to in-house (κcoefficient = 0.32, p = 0.02). Our study confirmed the presence and high prevalence of anti−CCHF antibodies in cattle based on three methods from all the five study districts, confirming presence and exposure of CCHFV. Given the zoonotic potential for CCHFV, we recommend a multidisciplinary public health surveillance and epidemiology of CCHFV in both animals and humans throughout the country.