Abstract Stable water isotope surveys have increasingly been integrated into river basins studies, but fewer have used them to evaluate impact of hydropower regulation. This study applies hydrologic and water isotope survey approaches to a Canadian Shield river basin with both regulated and natural flows. Historical streamflow records were used to evaluate the influence of three hydroelectric reservoirs and unregulated portions of the basin on downstream flows and changes in water level management implemented after an extreme flood year (1979). In 2013, water isotope surveys of surface and source waters (e.g., rainfall, groundwater, snowmelt) were conducted to examine spatial and temporal variation in contributions to river flow. Seasonal changes in relative groundwater contribution were assessed using a water-isotope mass balance approach. Within the basin, two regulated reservoirs exhibited inverted hydrographs with augmented winter flows, whereas a third exhibited a hydrograph dominated by spring snowmelt. In 2013, spatial variation in rain-on-snow and air temperatures resulted in a critical lag in snowmelt initiation in the southern and northern portions of the basin resulting in a dispersed, double peak spring hydrograph, contrasting with 1979 when a combination of rain-on-snow and coincident snowmelt led to the highest flood on record. Although eastern basin reservoirs become seasonally enriched in δ18O and δ2H values, unregulated western basin flows remain less variable due to groundwater driven baseflow with increasing influence downstream. Combined analysis of historical streamflow (e.g., flood of 1979, drought of 2010) and the 2013 water isotope surveys illustrate extreme meteorological conditions that current management activities are unable to prevent. In this study, the influence of evaporative fractionation on large surface water reservoirs provides important evidence of streamflow partitioning, illustrating the value of stable water isotope tracers for study of larger catchments.