Niphargus puteanus is the oldest described species of its genus and, in the past, was used as a taxonomic annotation for any subterranean amphipod record. For that reason, no clear knowledge exists about its actual range size and habitat preferences. We here applied a molecular taxonomic and phylogeographical approach based on mitochondrial and nuclear DNA to shed light on its distribution and to infer its demographic history. Furthermore, we analysed aquifer types and water flow regimes to provide a clearer picture of the species' ecological requirements. Our results indicate that N. puteanus is widely distributed north of the Alps, having its core range in the geomorphological natural region of the 'South German Scarplands' (SGS). Additionally, isolated satellite populations exist in the Taunus and the Sauerland, and two single individuals were collected in Luxembourg and in Austria, respectively. The species' maximal distribution range reaches 756 km between the two single-specimen records and 371 km within the SGS. A very high haplotype diversity was observed, revealing the presence of seven haplotype groups. All the haplogroups were present in the SGS and exhibited distinct spatial-genetic patterns. We thus inferred a high degree of population isolation, with the SGS being a potential long-term refugium for N. puteanus. Historical drift of specimens along major riverine networks (Rhine, Danube) may provide an evolutionary dispersal mechanism explaining the formation of satellite populations. From an ecological perspective, populations of N. puteanus were found to be closely associated with fast-flowing spring environments within fissured aquifers. This species should therefore be classified as both crenophile and stygophile, effectively exploiting resources of surface as well as subsurface spring waters.