In this study we are adding further information to phylogeography of Pipistrellus pipistrellus complex by discovery of isolated mitochondrial lineages in Europe, from the island of Crete (Greece) and central Mediterranean (Sicily, Corsica) and by adding new data about geographic distribution of clades within the complex. We performed phylogeographic study with aid of partial sequence of cytochrome b and with focus to the radiation centre of this group in the Mediterranean Basin. Within the clade P. pygmaeus s.l., we have discovered isolated lineage from Crete, which is sister taxon to P. hanaki from Libya. We have detected the occurrence of P. pygmaeus s.str. in northern Iran. In the clade P. pipistrellus s.l. we have discovered isolated cluster represented by populations from Sicily and Corsica, with phylogenetic relation to Moroccan lineage. This is a first evidence of separated phylogroup within the complex from central Mediterranean. We refer the occurrence of P. pipistrellus s.str. from the island of Cyprus. These data represent further arguments for importance of the Mediterranean region in phylogeny of the P. pipistrellus species complex. Proposed allopatric speciation scenario considers the role of environmental fragmentation during the Messinian Salinity Crisis, strengthen by preference of mountain habitats in Mediterranean populations. The species status of Cretan and central Mediterraenan forms is also discussed.