Summary1. Relationships between the assemblage structure of stygobionts (i.e. obligate groundwater species) and habitat conditions in aquifers were explored in the French Jura based on 16 environmental variables and presence/absence data on stygobiotic species. The two data sets were simultaneously collected at a total of 269 sites.2. The study aimed to (i) identify the environmental factors determining the composition of stygobiotic assemblages at the regional scale; (ii) define the ecological preferences of the collected stygobiotic species along environmental gradients; and (iii) describe species assemblages occurring in similar habitats. These aims were pursued with a view to refine approaches to groundwater biodiversity assessment and sampling strategies.3. A large number of stygobionts (62 species) were collected using a stratified sampling design. The measured environmental variables explained 72% of the overall variability in stygobiotic assemblage structure. Determining factors were primarily geology (pore size) and dissolved oxygen concentration, and secondarily altitude and distance of sampling sites from areas glaciated during the Pleistocene. Water chemistry and land-cover variables had little influence on the composition of stygobiotic assemblages.4. Ecological preferences of stygobiotic species were bounded along environmental gradients using Outlying Mean Index (OMI) analysis. Apart from rare species (frequency < 0.01), most stygobionts had a tolerance index >1, but residual tolerance observed in the most widespread species indicates sensitivity to other variables than those studied.5. Biodiversity hotspots mainly occurred in highly permeable geological formations such as karst and coarse alluvial environments with well oxygenated water situated between 200 and 500 m altitude. More intensive sampling of high-altitude sites is unlikely to yield additional species, but rare species may be found at low and mid altitudes and at sites far from the Würm glaciers.