Understanding the hydrology of large catchments has always been a major challenge due to the spatial heterogeneity of climate, geology, topography, soils and land use. However, precise knowledge of water cycling, storage and losses across large scale catchments is required to sustainably meet growing water demands and adjust water management strategies. This study used four large-scale, seasonal synoptic surveys in 2021 to characterize the spatio-temporal patterns of water isotopes (stable water isotopes and radioisotope tritium at natural abundance) to better understand the complex hydrology of the intensively managed 10,000 km2 catchment of the River Spree in Germany. Apart from the upper headwaters, the hydrology of the Spree is heavily regulated by reservoir releases, pumped minewater discharges, extensive wetlands and lakes, water abstractions and urban drainage. Moreover, the catchment is drought-sensitive with potential evapotranspiration (∼800 mm a year) often exceeding annual rainfall (∼600 mm). This is reflected in the isotopic composition of river water: In the steeper, upper headwaters, the river has a “flashy” rainfall-runoff response, with isotopes plotting close to the Global Meteoric Water Line (GMWL) consistent with dominant groundwater sources, but showing more influence by rainfall in winter. However, flows in the midstream parts are complex due to artificially enhanced baseflows and attenuated high flows from extensive reservoir releases and pumped groundwater releases from dewatering of mines. The reservoir waters are isotopically heavier and reflect the effects of open water evaporation. Fractionation effects strengthen downstream as managed wetland areas in the Spreewald and natural lakes further enhance evaporation and attenuate flows. Water abstractions for drinking water and to sustain the Oder-Spree canal reduce flows further. In Berlin, sources of urban runoff and waste water discharges reduce the effects of fractionation, though samples still plot below the meteoric water line. Seasonally, the effects of evaporation in the lower river network are strongest in summer and autumn, though they remain in winter and spring, indicating a large memory effect due to long mean travel times within the river system. Tritium variability along the river reflects inputs of younger and older water in different parts of the river system; though the influence of pumped groundwater means that the mean age of stream water (i.e., time elapsed since rainfall) in the lower river is likely to be >50 years. Climate change, together with increased population and economic growth in Berlin, is likely to increase pressures on the Spree system in future. In the coming years, the anticipated termination of the lignite mining will lead to a reduction in artificial inputs into surface water, which in turn may exacerbate water stress. We demonstrated that longer-term, synoptic isotope studies are valuable in better understanding the hydrology of such complex, large-scale, heavily modified river systems in terms of insights into water sources and mixing processes; thus, providing an evidence base for more sustainable management in the future.