Trypanosomatid infections of female horseflies were documented in three regions of Northwest Russia (Leningrad Oblast, Novgorod Oblast and the Republic of Karelia). The molecular analysis of four intestinal specimens and one culture isolated from the recta of infected horseflies revealed the presence of flagellates related to Trypanosoma theileri. This is the first registered case of such infection in the Northwestern Federal District and the first record of T. theileri-like trypanosomes in the vectors on the territory of the Russian Federation.