Aquatic biodiversity hotspots often emerge in regions with active tectonism, diverse climate conditions and complex basin configurations enabling episodic biotic isolation and exchange. The Anatolian microcontinent, located between the Mediterranean and Pontocaspian regions, has been considered a cradle of biodiversity for continental aquatic organisms. The Denizli Basin succession of SW Anatolia contains a “Didacna” mollusc fauna that could be the precursor of the modern Pontocaspian mollusc faunas of the Black Sea-Caspian Sea regions. However, the appearance of Pontocaspian faunas in the Denizli Basin and constraints upon their ages and dispersal pathways remain enigmatic. Moreover, the emergence of the Pontocaspian biota far into the Anatolian continental interior raises questions regarding the connectivity history and tectonic evolution of the Anatolian, Aegean and Pontocaspian realms. Here, we present an integrated stratigraphy of the ∼1 km thick succession of the Kolankaya Formation of the Denizli Basin, previously assigned to the Late Miocene. To date the first occurrence of Pontocaspian fauna in the Denizli Basin and to characterise accompanying palaeoenvironmental/palaeohydrological changes, we focus on three sets of approaches: dating (magnetostratigraphy and ⁴⁰Ar/³⁹Ar), biotic record (molluscs, ostracods and dinoflagellates) and hydrological connectivity (O- and C-isotopes and ⁸⁷Sr/⁸⁶Sr). We date the studied section as Early Pleistocene, spanning a time range of 2.6 Ma to 0.7 Ma. During that time, the Denizli Basin hosted an isolated to partially hydrologically open oligo-to mesohaline lake. The biotic record shows a drastic turnover of mollusc fauna from endemic Aegean-Anatolian and Pannonian/Paratethyan to Pontocaspian affinity at ∼1.8 Ma. The palaeogeographic evolution of the region, along with the geographically limited appearance of the Pontocaspian faunas, suggests a dispersal pathway from the Black Sea Basin via the Aegean Basin. Subsequently, a short incursion into the Denizli Basin may have occurred via a series of graben-type basins: either via the Söke-Milet Basin – Büyük Menderes Graben or via Izmir Bay – Gediz Graben. Our study shows that the Denizli Basin was not a cradle but rather a sink of the Pontocaspian biota during the Early Pleistocene. The new Early Pleistocene age assignment for the Pontocaspian fauna and the Kolankaya Formation in Denizli calls for a major reappraisal of models for the tectonic and stratigraphic evolution of SW Anatolia, including the regional interbasinal connectivity history.