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The black fly Simulium aureohirtum Brunetti (Diptera: Simuliidae) is geographically widespread and utilizes diverse habitats. Previous studies revealed high genetic diversity, suggesting that this species could be a species complex. In this study, we used mitochondrial cytochrome c oxidase II sequences to examine genetic structure and demographic history of S. aureohirtum in Thailand. High level of genetic variation was observed due to the existence of genetically divergent lineages (A, B, and C). Lineages A and C were geographically widespread, while lineage B was geographically restricted to the eastern part of Thailand. The geographic localization of lineage B suggested limitations in gene flow, which is most likely a result of ecologically different habitats. Large genetic differences between individuals of lineages A and C, but geographically sympatric distributions of the members of these lineages, are …