Background Trypanosoma evansi infects a large number of wild and domestic animals and causes a spoliative disease known as surra. It is mechanically transmitted, mainly by biting flies of the genera Tabanus and Stomoxys. The detection of T. evansi DNA in the feeding apparatus of Dichelacera alcicornis and Dichelacera januarii from South America is reported, to the best of our knowledge, for the first time. Methods Tabanids were collected weekly from February 2018 to February 2019 from two sites. The feeding apparatus was removed and DNA extraction, polymerase chain reaction and sequencing were performed. Results A 205-base pair fragment of the variant surface protein RoTat 1.2 gene, confirmed by DNA sequencing, was amplified from the feeding apparatus of D. alcicornis and D. januarii. Conclusions This is, to the best of our knowledge, the first record of T. evansi DNA in South American tabanids. Graphical abstract