A short segment (135 bp) of the control region and a partial sequence (394 bp) of the 12S-rRNA gene in the mitochondrial DNA of Crocidura russula were analyzed in order to test a previous hypothesis regarding the presence of a gene flow disruption in northern Africa. This breakpoint would have separated northeast-African C. russula populations from the European (plus the northwest-African) populations. The analysis was carried out on specimens from Tunisia (C. r. cf agilis), Sardinia (C. r. ichnusae), and Pantelleria (C. r. cossyrensis), and on C. r. russula from Spain and Belgium. Two C. russula lineages were identified; they both shared R2 tandem repeated motifs of the same length (12 bp), but not the same primary structure. These simple sequence repeats were present in 12-23 copies in the right domain of the control region. Within the northeast-African populations, a polymorphism of repeat variants, not yet found in Europe, was recorded. A neighbor-join tree, which was built by sequences of the conserved 12S-rRNA gene, separated the two sister groups; it permitted us to date a divergence time of 0.5 Myr. Our data discriminated two different mitochondrial lineages in accordance with the previous morphological and karyological data. Ecoclimatic barriers formed during the Middle Pleistocene broke the range of ancestral species in the Eastern Algeria (Kabile Mountains), leading to two genetically separate and modern lineages. The northeast-African lineage can today be located in Tunisia, Pantelleria, and Sardinia. The northwest-African lineage (Morocco and West Algeria), reaching Spain by anthropogenic introduction, spread over north Europe in modern times. The Palaearctic C. russula species is monophyletic, but a taxonomical revision (ie, to provide a full species rank for the northeast taxa and to put in synonymy some insular taxa) is required.