Diversification and speciation processes in the endemic Anatolian Aphanius taxa are well documented today based on crossbreeding experiments and molecular data. On the other hand, otolith characteristics have rarely been utilized to demonstrate these processes in Aphanius. In this study, otoliths of 250 specimens of Aphanius anatoliae, A. asquamatus, A. danfordii, and A. villwocki were studied, mainly by SEM. The otoliths display morphological characters that clearly discriminate the four species. The otoliths were found to be morphologically different in five out of ten populations of A. anatoliae anatoliae. However, the differences in otolith morphology between subspecies of A. anatoliae are less obvious. Variation of the general otolith variability is documented in two of the four A. danfordii populations, and may reflect differences in the genetic diversity. The results obtained from otoliths correspond in part with previous reports on zoogeographic groups and phylogenetic clades.