The present study investigates morphologically similar species in the amphipod genus Niphargus, with special emphasis on three presumably closely-related species: N. fontanus, N. aquilex, and N. schellenbergi. The distribution ranges of these species overlap in Central Europe, and ambiguity of the current diagnostic characters has likely resulted in misidentifications. To address this issue, we carried out molecular-phylogenetic analyses of Cytochrome Oxidase Subunit 1 gene (COI) and 28S rDNA sequences for 34 individuals of the three species from 27 sites in Central Europe and Great Britain. We also included 14 GenBank sequences for two additional species, N. virei and N. rhenorhodanensis, to increase the resolution potential among our in-group taxa. In addition, about 100 specimens of the three species were examined for a reappraisal of diagnostic characters. Representative specimens taken from various subterranean habitat types were examined for each of the three species. A new morphological key, utilizing improved characters, is presented to clearly distinguish between N. fontanus, N. aquilex, and N. schellenbergi. Molecular-phylogenetic analysis using COI sequence data indicates geographically well-delimited clades for N. aquilex and N. fontanus. Our data suggest that some previously recorded niphargids from the Harz Mountains of Germany, initially identified as N. fontanus or N. aquilex, were all N. schellenbergi.