In this study, chromosomal studies were carried out on four species of the genus Aphanius (Osteichthyes, Cyprinodontidae). Metaphase chromosomes were obtained from kidney cells. The diploid chromosome numbers of Aphanius anatoliae, A. danfordii, A. splendens and A. villwocki were 2n=48, and consisted of three pairs of submetacentric and 21 pairs of subtelocentric chromosomes. The arm number (NF) was 54. Constitutive heterochromatin regions were observed on the centromeres of chromosomes in all four species by using C-banding. Nucleolus Organizer regions (NOR) were observed on the short arms of two pairs of chromosomes. It is believed that the data obtained will make a contribution to fish cytogenetics.