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Background: Tabanus bromius (Diptera: Tabanidae) is one of the most notable Tabanidae species of veterinary and medical importance distributed throughout the Palearctic region. In this study, we investigate the genetic diversity and the phylogeographic structure of T. bromius sampled from Turkey, Croatia, and Iran., Methods: For this purpose, a 686-base-pair (bp) fragment of mitochondrial DNA cytochrome oxidase I gene (COI) and 1339 bp of the nuclear DNA internal transcribed spacer (ITS) were sequenced from 247 individuals representing 15 populations., Results: The sequences generated 169 COI haplotypes and 90 ITS alleles. A higher haplotype/allele diversity (h = 0.9909 for the COI gene and Ad = 0.8193 for the ITS region) compared to a low nucleotide diversity (π = 0.020605 for COI gene and π = 0.013667 for the ITS region), present for a high number of singleton and private haplotypes/alleles imply population expansion in the past. The results of phylogenetic analysis led to the uncovering of geographically significant groupings of lineages with regard to the entrance of the species into Anatolia and the location of major geographic barriers. According to current data, the species appears to have entered Turkey from Caucasia and Iran. A molecular clock applied to the COI data suggests that T. bromius diverged from the outgroup species nearly 8.83 million years ago, around the end of the Miocene era., Conclusions: The results of this study indicate remarkable genetic diversity across the studied range of the species. High haplotype/allele versus low nucleotide diversity and demographic analyses implied that the T. bromius populations have undergone a series of expansions and retreats in the past. Our current findings suggest that T. bromius split from outgroups around the Late Miocene. Subsequent diversification events during the climatic and environmental fluctuation times of the Late Pliocene and Early Pleistocene periods also significantly influenced the species, resulting in the formation of some major genetic lineages. The phylogenetic analyses indicate that T. bromius most likely entered Turkey from the Caucasus region and Iran., (© 2021. The Author(s).)