We analysed the phylogenetic relationships between M. d. daubentonii and M. d. nathalinae based on 1,010 bp of the cytochrome b mtDNA gene. The inference based on molecular phylogenetics methods shows that these two morphotypes correspond to two mitochondrial groups within the Iberian Peninsula. Our results also support the model of ‘refugia within refugia’, where M. d. daubentonii has spread north and M. d. nathalinae has became an Iberian endemism. The haplotype network indicates haplotype sharing between Monfurado and S. Mamede (Portugal) and Bavaria (Germany), demonstrating current or recent dispersal and gene flow between these populations. Myotis d. nathalinae displays a substructure showing that populations under the same climate type are more related. As a distinct Iberian endemism, the conservation status of M. d. nathalinae should be reviewed.