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The purpose of this study was to determine the phylogeographic structure of the brackish-hypersaline cyprinodont fish Aphanius fasciatus (Valenciennes, 1821), using sequencing and RFLP analysis of a 1,330 bp mitochondrial DNA segment containing part of the 16S rRNA gene as well as the genes for tRNA-Leu, NADH subunit 1 and tRNA-Ile. Individuals were collected from 13 different sites in Greece and Turkey, while seven published A. fasciatus sequences were also included to cover the area of distribution of the species. Pairwise sequence divergence values ranged from 0 to 4.51%. Congruent phylogenies were recovered with maximum likelihood, maximum parsimony and neighbour-joining methods. All analyses revealed two main groups. The first group consists of populations from almost all localities that drain into the Aegean Sea. The second group comprises the remaining population samples, which in some cases seem to consist of population-specific subgroups. Our results show that vicariant events have predominantly affected the evolution of A. fasciatus, with the Messinian salinity crisis having shaped the present genetic structure of its populations. Additionally, the life-history traits of the species, which determine a low potential for dispersal, coupled with the typical fragmentation of brackish-hypersaline water habitats have led to a high degree of isolation of A. fasciatus populations, even at restricted spatial scales. Analysis of the partitioning of the total amount of polymorphism with analyses of molecular variance (AMOVA) gave a value of F ST = 84.6%. Potential conservation policies concerning A. fasciatus should also consider the low-genetic variability in the majority of its populations and the presence of fixed haplotypes in some of them.