To explain the origin of the differentiation of the spring-inhabiting fauna on an island system, this study focused on the Aegean Islands and the Bythinella snails as a model organism. We inferred the phylogeographic pattern of the Aegean Bythinella with two molecular markers, and compared the inferred pattern with the geological history of the region and the estimated levels of biodiversity of Bythinella in Greece. 95 sequences of COI and 60 of ITS-1 were obtained from samples collected from Andros, Crete, Naxos, Chios, Kithira, and western Turkey, and pooled with previously published sequences from continental Greece. Phylogenetic analysis based on the COI revealed seven new clades. On Crete, three distinct clades were identified. The estimated divergence time indicated that the geological history of the island, which was divided into smaller islands until 2-3 Mya, was most probably responsible for such high diversity. Bythinella populations inhabiting Turkey were distinct from the Aegean populations, and the divergence began after the Zanclean flood (5.3 Mya). Shell morphology and anatomy of reproductive organs confirmed that molecularly distinct clades were real entities. Biodiversity analysis revealed that, in addition to five previously known Bythinella hotspots, there is a sixth in central Greece.Keywords Origin and history of spring fauna Á Springs as ephemeral habitats Á mtDNA Á COI Á ITS-1 Á Morphology Á Phylogeography Á Biodiversity