In this study, a comparative analysis of the vertebral column and the caudal skeleton of 245 specimens of 10 Aphanius and three Aphaniops species was conducted based on X-ray imaging, and interspecific variation of these characters was examined. The vertebral bending index showed that straight and almost straight vertebral columns were more common in Aphaniops than in the Aphanius species. The numbers of abdominal and caudal vertebrae, principal caudal fin rays, and principal rays supported by the hypural plate were significantly lower in Aphaniops than in Aphanius species. Those species of Aphanius which were found in higher latitudes or altitudes had more vertebrae than the remaining Aphanius species and all members of Aphaniops, revealing the role of environmental factors. The number of preural (PU) vertebrae and the width of neural and haemal spines of preural vertebrae 2-4 were significantly higher in Aphaniops than in the Aphanius species. The conspicuous variations detected among localities of A. arakensis highlight the importance of more profound studies on the diversity of this species. In the genus Aphaniops, 17% of specimens showed a straight epural bone and 83% showed a sinusoidal bone. In comparison, Aphanius species showed that 91% and 7% of specimens displayed straight and sinuous shape epural bone, respectively. Such intra-species polymorphism of the epural character state was first reported for Aphaniid species. Hence, the previously proposed synapomorphy for the genus Aphaniops based on sinusoidal epural bones may need further investigation.