We use two mitochondrial DNA fragments with different substitution rates (cytochrome b gene and the control region) to address the following phylogeographic questions about western Palaearctic populations of the barbastelle bat (Barbastella barbastellus): 1) Do the Iberian populations of barbastelles show any genetic discontinuity associated with its present fragmented distribution?, 2) Is the Gibraltar Strait an effective barrier to gene flow for barbastelles? and 3) Is the subspecies from the Canary Islands genetically distinct from continental barbastelles? Our molecular survey shows that there is only a shallow genetic structure among populations of the Iberian Peninsula and Morocco, and probably, even across Europe until Thrace, although this last point needs to be confirmed. The Gibraltar Strait has not played any significant role as a biogeographic barrier to prevent the recent passage of European barbastelles to Morocco (or vice versa). Our phylogenetic reconstructions also confirm the taxonomic distinction of B. barbastellus guanchae as an endemic subspecies confined to the Canary Islands. The precise origin of this Canarian taxon is, nevertheless, still unclear as its mitochondrial lineage is distinct from any lineage found so far in Morocco and Iberia. This important genetic distinctness suggests either a relatively ancient colonization of the Canary Islands or that the source population of the founders have not yet been identified.