Mosquitoes belonging to the Aedes mariae complex, including Aedes mariae (Sergent & Sergent), Aedes zammitii (Theobald), and Aedes phoeniciae (Coluzzi & Sabatini), are among the few animals inhabiting the extreme habitat of sea rock pools. Morphological analysis of these species and crossing experiments conducted in laboratory and natural conditions inferred the occurrence of three taxa with a parapatric distribution along the coasts of the Mediterranean Basin. To date, however, genetic characterization of the three species within the Ae. mariae complex and an assessment of their interspecific differentiation are lacking. In this study, we used both nuclear (i.e., 14 allozymic loci) and mitochondrial genetic markers (i.e., sequences of the cytochrome oxidase I and II genes) to genetically characterize the three species belonging to the complex. Then, we used mitochondrial markers to infer phylogenetic relationships between the species and estimate the time to most recent common ancestor (TMRCA). The allozymic and mitochondrial markers showed the occurrence of three distinct gene pools, namely, Ae. mariae, Ae. zammitii, and Ae. phoeniciae. The TMRCAs for the entire in-group were estimated to have occurred during the early Pleistocene (i.e., mean node age of 1.739 million years ago). An important role of Pleistocene climatic changes could be suggested in the origin of the species of the Ae. mariae complex and in shaping their pattern of intraspecific genetic diversity.