Freshwater snails that inhabit islands are excellent model organisms for testing relationships between geological events and phylogeography, especially in the Aegean region. Although many Aegean islands were searched in the present study, species of the genus Pseudorientalia were only found on Lesvos, Samos, and Chios. Phylogenetic relationships between specimens living on these three islands were analysed using COI and 16S rRNA molecular markers and morphological data. A high level of diversity was found between islands. Genetic distances between clades showed differences high enough for the samples from different islands to be considered distinct species (p-distance: 0.105–0.133). These results are also supported by obvious morphological differences in shell morphology between islands. The mean divergence time between the Lesvos clade and Samos/Chios clade was 24.13 ± 3.30 Mya; between the Samos and Chios clades the divergence time was 14.80 ± 1.11 Mya. Our data suggest that high divergence may have occurred between Pseudorientalia populations during the Upper and Middle Miocene, when the Aegean region was part of a united landmass. It is possible that the observed highly divergent Pseudorientalia clades are relicts of high regional diversity that existed in the past.