Ancient lakes harbour endemic faunas that fascinated biologists for centuries. Their size, relative stability, and spatial isolation over geological time scales promoted speciation and ecological diversification within their narrow confines, turning these water bodies into convenient natural laboratories for evolutionary studies. The Caspian Sea is the world’s largest ancient lake and, together with the Aral, Azov and Black seas, forms the Ponto-Caspian region – both an endemicity hotspot and a significant donor area of invasive species. These water bodies are remnants of the largest lake in the geological record, the Paratethys Sea, which underwent significant environmental change during the last 15 million years. Amphipod crustaceans attained the highest diversity of any Ponto-Caspian organism and, due to human intervention, up to 40% of these species have now expanded well beyond the native region, some becoming notorious invaders. However, their temporal origin and subsequent diversification are poorly understood and subject to debate due to methodological biases, as well as poor taxonomic and molecular data coverage. Therefore, in this project we aim to unravel the evolutionary history of Ponto-Caspian amphipods at an unprecedented taxonomic and temporal resolution by integrating for the first time dense taxonomic sampling, multiple DNA markers, morphology, and fossils with state-of-the-art molecular dating methods. The project results will bring crucial insight into our understanding of evolution in ancient lakes and basins, origin of invasive species, and illuminate the tumultuous geological past of the Ponto-Caspian region.