Monitoring of terrestrial and aquatic species assemblages at large spatial scales based on environmental DNA (eDNA) has the potential to enable evidence-based environmental policymaking. The spatial coverage of eDNA-based studies varies substantially, and the ability of eDNA metabarcoding to capture regional biodiversity remains to be assessed; thus, questions about best practices in the sampling design of entire landscapes remain open. We tested the extent to which eDNA sampling can capture the diversity of a region with highly heterogeneous habitat patches across a wide elevation gradient for five days through multiple hydrological catchments of the Swiss Alps. Using peristaltic pumps, we filtered 60 L of water at five sites per catchment for a total volume of 1800 L. Using an eDNA metabarcoding approach focusing on vertebrates and plants, we detected 86 vertebrate taxa spanning 41 families and 263 plant taxa spanning 79 families across ten catchments. For mammals, fishes, amphibians and plants, the detected taxa covered some of the most common species in the region according to long-term records while including a few more rare taxa. We found marked turnover among samples from distinct elevational classes indicating that the biological signal in alpine rivers remains relatively localised and is not aggregated downstream. Accordingly, species compositions differed between catchments and correlated with catchment-level forest and grassland cover. Biomonitoring schemes based on capturing eDNA across rivers within biologically integrated catchments may pave the way toward a spatially comprehensive estimation of biodiversity.