Among the species of Aphanius Nardo, 1827, Aphanius dispar (Rüppell, 1828) is the most common taxon and has long been viewed as representing a species group rather than a single species. This study provides comprehensive data on the phylogenetic relationships, morphology, and otoliths within the A. dispar species group, including the description of a new species. Our data demonstrate that the “true” A. dispar is restricted to the Red Sea drainages and that all other populations hitherto identified as A. dispar actually represent separate species. Four main clades are defined and named for the geographic areas in which the respective species of Aphanius occur. The oldest one is the “Red Sea clade,” it comprises A. dispar. The “Dead Sea clade” is represented by A. richardsoni (Boulenger, 1907). It is sister to both the “Hormuzgan clade” in S Iran (containing A. hormuzensis sp. nov. and A. ginaonis (Holly, 1929)) and the “Persian Gulf & Gulf of Oman clade” (comprising A. stoliczkanus (Day, 1872)). The species separation within the A. dispar group is confirmed by the distinctive otolith morphology of each species. Moreover, we present a time-calibrated phylogeny (chronogram) for the A. dispar species group using †A. princeps (16–17 Mya) as a minimum age and the first appearance of †Prolebias (33–34 Mya) as a maximum age for the genus Aphanius. The evolution and historical biogeography routes are discussed based on the outcome of the chronogram and in the context of the geological and climatic history of the Near East in Pliocene–Pleistocene times.