Wesselsbron is a neglected, mosquito-borne zoonotic disease endemic to Africa. The virus is mainly transmitted by the mosquitoes of the Aedes genus and primarily affects domestic livestock species with teratogenic effects but can jump to humans. Although no major outbreak or fatal case in humans has been reported as yet worldwide, a total of 31 acute human cases of Wesselsbron infection have been previously described since its first isolation in 1955. However, most of these cases were reported from Sub-Saharan Africa where resources are limited and a lack of diagnostic means exists. We describe here two molecular diagnostic tools suitable for Wesselsbron virus detection. The newly established reverse transcription-quantitative polymerase chain reaction and reverse-transcription-recombinase polymerase amplification assays are highly specific and repeatable, and exhibit good agreement with the reference assay on the samples tested. The validation on clinical and veterinary samples shows that they can be accurately used for Wesselsbron virus detection in public health activities and the veterinary field. Considering the increasing extension of Aedes species worldwide, these new assays could be useful not only in laboratory studies for Wesselsbron virus, but also in routine surveillance activities for zoonotic arboviruses and could be applied in well-equipped central laboratories or in remote areas in Africa, regarding the reverse-transcription-recombinase polymerase amplification assay.