Anaplasma marginale is transmitted biologically by infected ticks or mechanically by biting flies and contaminated fomites. In tick-free areas, such southern Uruguay, horseflies could be the principal vectors of this pathogen to bovines, causing anaplasmosis occurrence. Therefore, the objective of this work was to detect the presence of A. marginale by MSP-5 PCR and Sanger sequencing in the most prevalent species of horseflies using different collection methods in the localities of Colonia, Taquarembó and Paysandú, Uruguay. Eight horsefly species were tested (Dasybasis missionum, Poeciloderas lindneri, Tabanus campestris, T. claripennis, T. fuscofasciatus, T. platensis, T. tacuaremboensis and T. triangulum); four species were found positive for A. marginale, with D. missionum and P. lindneri having the most frequent infections, while only one individual each of T. fuscofasciatus and T. tacuaremboensis was positive. Both D. missionum and P. lindneri were positive for A. marginale in tick-free areas, with implications that are discussed in this report.