Aim The purpose of this study is to (1) investigate the Quaternary history of Lake Pamvotis using fossil ostracod faunal and isotopic analyses, and (2) use this fossil evidence to assess the pedigree of the lake’s fauna and place it within a regional palaeobiogeographical context.Location Lake Pamvotis, situated in the Ioannina Basin, north-western Greece.Methods Fossil samples were recovered from a 319 m-long, 500 ka core drilled on the eastern shore of the current lake. Stable isotopic analyses were carried out on ostracod shell calcite.Results A total of 17 ostracod taxa were recovered, of which 13 occur in the modern lake. Fossil assemblages and corresponding isotopic profiles from the Last (Eemian) Interglacial and Last Glacial-Holocene transition are presented and compared with modern-day data. Some taxa (e.g. Leptocythere sp. A) appear to be lake basin endemics.Main conclusions The ostracod fauna is typical of temperate, carbonate-rich standing waters and includes both European and Balkan endemic elements. The fossil fauna, coupled with the isotopic data, provides evidence for past lake-level change, including a progressive deepening throughout much of the Last Interglacial and a gradual shallowing throughout the subsequent glacial. This culminated in a distinct interval of low lake-levels at the height of the last glaciation. The Lateglacial witnessed a partial recovery in lake depth, although shallowing occurred once again during the Holocene. The biogeographical affinities of the ostracod fauna, whilst not unequivocal, nevertheless suggest that speciation in the region has been by vicariance and hydrological isolation, probably within the Pleistocene.