To date, 28 species of Aphanius have been recognized in the World, of which 14 species are described from six endorheic and eight exorheic basins in Iran. Here we report the taxonomic status and phylogenetic relationships of the 14 Iranian Aphanius species to understand their evolutionary history, and thus to contribute to future conservation strategies and habitat management. Based on the cytochrome b gene in combination with geological data, they are representatives of three large clades i.e. (i) A. dispar clade (A. dispar, A. ginaonis, A. furcatus and A. sirhani), (ii) A. mento clade and (iii) Inland Aphanius species clade (A. vladykovi, A. darabensis, A. shirini, A. isfahanensis, A. farsicus, A. arakensis, A. kavirensis, A. mesopotamicus, A. pluristriatus and A. sophiae). The latest clade comprises three subclades (A. vladykovi-, A. shirini- and A. sophiae subclade) and represents an “old” evolutionary group that had diverged in the Late Miocene and Early Pliocene (ca. 10–5 Million years ago) (A. vladykovi and A. shirini subclades and A. isfahanensis of the A. sophiae subclade) and a “young” evolutionary group that may have diverged during the Late Pleistocene (100,000–11,700 y. ago) and Early to Middle Holocene (11,700–4,000 y. ago) (A. sophiae subclade, but without A. isfahanensis). The A. sophiae subclade is the most remarkable group with seven species that they are superficially very similar and often not clearly distinguishable by external characters alone. However, differences in cytochrome b and also between the otoliths clearly separate all species of this subclade. In addition, the species of the A. sophiae subclade show widely separated distribution areas with no hydrological connectivity in between, which additionally confirm their clarification as distinctive species. All Iranian Aphanius species are native except A. dispar, A. mesopotamicus and A. mento.