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Black flies (Diptera: Simuliidae) are well known as annoying biting pests and vectors of disease-causing agents to humans and other animals. Despite their relevance, no studies have been conducted on Hispaniola for decades. Our work aimed to determine the species composition, relative abundance, breeding sites, phylogenetic relationships, and biting habits of black flies from an important ecotourism spot in La Vega Province (Dominican Republic). Simulium ochraceum sensu lato Walker, Simulium haematopotum Malloch, and Simulium quadrivittatum Loew were identified by both morphological and molecular analyses. Black flies were abundant in most (92% of the total) of the flowing water courses studied and their relative abundance varied depending on the method of collection and type of water course. Simulium haematopotum was prominent in large rivers whereas the other two black fly species were …