Within the Cirolanidae, a widespread family of marine isopods, about 23 genera are stygobitic and inhabit phreatic and anchialine ecosystems, with many endemic species. The Mediterranean area has a high biodiversity of subterranean cirolanids, which are considered thalassoid limnostygobionts. A molecular analysis was conducted using mtDNA genes to infer the phylogeny of species belonging to six of the seven stygobitic genera of Cirolanidae inhabiting the Mediterranean basin and to two American taxa: Faucheria faucheri, Marocolana delamarei, Saharolana seurati, Sphaeromides virei virei, Turcolana sp., 13 taxa of the genus Typhlocirolana and two American species, Antrolana lira and Speocirolana bolivari. The Typhlocirolana species are widespread in the western Mediterranean basin, with a concentration of taxa in the Maghreb region. Turcolana sp. is localised in the eastern Mediterranean, while F. faucheri and S. v. virei are north Mediterranean taxa. S. seurati, the taxon least morphologically adapted to subterranean life, belongs to a monospecific genus present in a Tunisian spring. The molecular phylogeny showed a high affinity among the American taxa and the Mediterranean Sphaeromides, clustering in the Sphaeromides group identified by previous morphological studies. Typhlocirolana species and M. delamarei constitute their sister clade within the Sphaeromides group. F. faucheri appears to be a sister clade of the Sphaeromides group. S. seurati, showing reduced troglobitic adaptations, assumes disparate and unsolved positions in the phylogenetic reconstructions. The molecular data suggest that a combination of vicariance and dispersal events, occurring from 180 to a few million years ago, combined to bring about the present distribution pattern of Mediterranean cirolanid isopods.