A new killifish species, Aphanius isfahanensis, is described from the Isfahan basin of Iran. It is distinguished from the other Iranian species of Aphanius by a suite of overlapping but significantly different meristic and morphometric characters, adult color pattern and molecular character states of homologous mitochondrial DNA sequences. Based on the phylogenetic analysis of molecular sequence data, the new species is hypothesized to be sister taxon to A. sophiae plus A. persicus, which also occur in Iran
