Understanding spatial patterns of biodiversity within the context of long‐term climatic shifts is of high importance, particularly in the face of contemporary climate change. In comparison to aboveground taxa, subterranean organisms respond to changing climates with generally much lower dispersal and recolonization potential, yet possible persistence in refugial groundwater habitats under ice‐shields. However, knowledge on general and geographically large‐scale effects of glaciation on contemporary groundwater biodiversity patterns is still very limited. Here, we tested how Late Pleistocene glaciation influenced the diversity and distribution of 36 groundwater amphipod species in Alpine and peri‐Alpine regions, characterized by extensive glaciation cycles, and how its legacy explains contemporary diversity patterns. We based our analysis on an unprecedented density of ~ 1000 systematic sampling sites across Switzerland. Using presence–absence data, we assessed biodiversity and species' ranges, and calculated for each site within‐catchment distance to the Last Glacial Maximum (LGM) glacier extent. We then applied a sliding window approach along the obtained distance gradient from LGM ice‐covered to ice‐free sites to compute biodiversity indices reflecting local richness, regional richness, and differentiation, respectively. We found a strong signal of the LGM ice extent on the present‐day distribution of groundwater amphipods. Our findings revealed pronounced species turnover and spatial envelopes of individual species' occurrences in formerly ice‐covered, ice‐free, or transitional zones, respectively. While local richness remained constant and low along the LGM distance gradient, groundwater communities in LGM ice‐covered areas were more similar to each other and had lower gamma diversities and decreased occurrence probabilities per sliding window compared to communities in Pleistocene ice‐free areas. These results highlight the significant impact of Pleistocene glaciation on shaping biodiversity patterns of subterranean communities and imprinting contemporary distribution of groundwater organisms.