The subterranean Isopods belonging to the genus Stenasellus have an interesting disjunct distribution in the peri-Tyrrhenian area with morphologically closely related taxa occurring in Sardinia, Corsica, Tuscany and in the Pyrenees phreatic and interstitial waters. Because the dispersal capacities of these organisms are limited, their distribution has been associated traditionally with the tectonic events leading to the separation of the Sardinia–Corsica microplate from the Pyrenees and its subsequent movement towards the Italian peninsula. We sequenced a fragment of the mtDNA cytochrome oxidase I gene (COI) for multiple populations of the S. racovitzai species-group (Corsica, Sardinia, Tuscany) and S. virei (Pyrenees). We found that multiple phylogenetic analyses always gave the same topology, which is consistent with the genetic relations found using allozyme data, and with the palaeogeography of the area. The molecular data suggest that a combination of vicariance and dispersal events explain most effectively the present distribution pattern of these organisms. We also calculated COI rates and calibrated them against absolute time, taking advantage of the availability of two geologically based time estimates. Rates on all substitutions are similar to those published for other crustaceans for the same COI fragment, including taxonomically and ecologically distant groups. Rates on third codon positions or on transversions are generally lower than those found in other crustaceans.