•Microsatellite markers for five surface and groundwater species.•Populations of surface and groundwater species are highly variable and structured.•Neighboring populations share low levels of gene flow.•Genetic methods reveal hidden natural or man-made groundwater hydrological patterns.•The StygotracingⓇapproach represents a promising tool for drinking water management. Groundwater is one of our most important resources, however groundwater ecosystems are among the most understudied habitats of the planet earth. Studies on groundwater organisms are hampered by the difficult accessibility of species, the lack of morphological differentiation and the limitation for laboratory cultures. One important approach to overcome these shortcomings is to provide sensitive genetic methods to unravel patterns of biodiversity, population structure and gene flow in natural populations. In this study we present five sets of microsatellite markers developed for the isopods Asellus aquaticus and Proasellus slavus, the cyclopoides Paracyclops fimbriatus and Acanthocyclops sensitivus and the harpacticoide Bryocamptus echinatus (Crustacea). Two of these species were subjected to detailed population genetic analyses: We studied 501 specimens of Asellus aquaticus from four different regions in Northern Germany using nine microsatellite markers and 70 specimens of Bryocamptus echinatus using nine microsatellite markers from three different sampling sites in South-Western Germany. Our results show that genetic diversity is high (A. aquaticus: 10 to 20 and B. echinatus: 4 to 18 alleles per locus) among populations of aquatic invertebrates, populations are highly differentiated (FST > 0.2) and genetic differentiation was associated with geographic patterns. Applications of molecular genetic methods and their use for the detection of hydrological exchange processes relevant for drinking water suppliers are demonstrated and discussed.