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Purpose: The species of the genus Trypanosoma are carried and transmitted by horseflies parasitizing a high diversity of vertebrates. In the Coastal Plain of Rio Grande do Sul, southern Brazil, Tabanus triangulum is the most abundant species and, similarly to the other species of horseflies, there is little knowledge about its vector competence. Therefore, this study aimed to screen the field-collected T. triangulum for the presence of Trypanosoma, to estimate infectivity., Methods: Horseflies were sampled by the Malaise trap in the forest fragments at the coastal plain and DNA was extracted from whole body flies. The Polymerase Chain Reaction was performed., Results: Horseflies presented amplification of 18S ribosomal gene-specific of Trypanosoma species. DNA sequencing and phylogenetic analysis positioned the strains in the Kaiowa clade with Trypanosoma kaiowa, associated with the crocodilian clade of Trypanosoma., Conclusion: This study represents the first report of the presence of the Tr. kaiowa in T. triangulum and the expansion of the parasite's range further south in South America., (© 2021. Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)