A substantial number of species within the tooth-carp Aphanius Nardo, 1827 (Cyprinodontidae, Teleostei) has been recorded from the endorheic drainage systems of Iran, and several isolated populations in these systems may deserve species status. Descriptions of these species and populations have been based mainly on morphological and molecular data; however, the characters related to the fish scales have not up to now been intensively studied and employed for the identification of the species belonging to this genus. The objective of this study is to test as to whether (i) scale surface morphology, (ii) scale surface microstructure, and (iii) scale size can be used to discriminate species and/or populations and, (iv) to discuss the possible causes of the observed differences. To achieve these objectives, scales of three species of the genus Aphanius from endorheic basins in SW Iran, viz. A. sophiae (nine populations from the Kor River Basin), A. farsicus (four populations from the Maharlu Lake Basin) and A. pluristriatus (a single population from the Mond River Basin) have been studied using SEM images, scale measurements, and uni- and multivariate statistics. It is opined that scale surface morphology and microstructure cannot help in distinguishing the species, but can be employed to discriminate certain populations of A. sophiae (those from Safashahr, Kharameh, Tashk, Gol). In addition, scale size and J-indices, respectively, represent a valuable tool for species separation, which corroborates earlier studies for the use of these indices in taxonomy. Major driving forces of the differentiation within A. sophiae probably include habitat fragmentation resulting from the geological history and local adaptations. Thus the differentiation results from a balance between both genetic and environmental effects.