We recovered molecular phylogenetic relationships amongst species of the genus Ablepharus in Iran and Iraq. Partial sequences of three mitochondrial genes (cytochrome C oxidase subunit I – COI, 12S rRNA and 16S rRNA) were analysed. In addition, phylogenetic relationships and taxonomic evaluation of Ablepharus species in Cyprus, India, Greece, Turkey and Syria were performed using partial sequences of the 16S rRNA gene. Phylogenetic trees and estimated genetic distances showed that the Ablepharus populations of Iran and Iraq clustered into three distinct clades. One is found in northwest Iran (A. bivittatus in Ardabil, East and West Azerbaijan and Hamedan Provinces). The second clade, formed by A. chernovi, is found only in Uromia. The third and most heterogeneous clade is divided into two subclades, the first includes two lineages of Ablepharus in Khorasan Razavi and Semnan Provinces (A. pannonicus) and in eastern and south-eastern Iran (A. grayanus); the second subclade is distributed in the eastern part of Iraq and west and south-western Iran (Ablepharus sp.). Our analyses indicated that splitting of A. chernovi within the genus occurred in the early Miocene [about 22.5 million years ago (Mya)]. Ablepharus bivittatus diverged 15.2 Mya, in the middle Miocene. Ablepharus pannonicus diverged in the late Miocene (8.4 Mya) and A. grayanus separated in the late Miocene (6.7 Mya). The lineages of eastern Iraq and south-western Iran (Ablepharus sp.) diverged also in the late Miocene (7.0 Mya).