We studied genetic divergence in a group of exclusively stygobiont isopods of the family Stenasellidae. In particular, we assessed evolutionary relationships among several populations of Stenasellus racovitzai and Stenasellus virei. To place this study in a phylogenetic context. we used another species of Stenasellus, S. assorgiai, as an outgroup. S. racovitzai occurs in Corsica, Sardinia and in the fossil islands of the Tuscan Archipelago, while S. virei is a polytypic species widely distributed in the central France and Pyrenean area. This vicariant distribution is believed to be the result of the disjunction of the Sardinia-Corsica microplate from the Pyrenean region and its subsequent rotation. Since geological data provide time estimates for these events, we can use the genetic distance data to calibrate a molecular clock for this group of stygobiont isopods. The calibration of the molecular clock reveals a roughly linear relationship (r = 0.753) between the genetic distances and absolute divergence times, with a mean divergence rate (19.269 Myr/DNei) different from those previously reported in the literature and provides an opportunity to shed some light on the evolutionary scenarios of other Stenasellus species.