Isotope hydrograph separation is a powerful tool to investigate catchment functioning. In mosthydrograph separation studies, a pre‐event baseflow sample is used to represent the pre‐event water, andthus, baseflow is assumed to be a mixture of all the water that is stored in the catchment. However, baseflowmay not be representative of all water stored in the catchment because some sources may not contribute tobaseflow. This is problematic when the isotopic composition of the sources is highly variable. Wequantified the effects of spatial variability in the shallow groundwater isotopic composition on pre‐eventwater characterization and hydrograph separation results. We compared the composition of groundwatersampled at 38 wells in a 0.2 km2pre‐alpine catchment with stream water sampled before, during, and afterthree rainfall events. We estimated the number of groundwater samples needed to characterize the averagegroundwater composition in the catchment and its spatial variability and compared the results oftwo‐component hydrograph separations for different ways to characterize the pre‐event water. We foundthat differences in the calculated pre‐event water fractions and uncertainties were large and depended onwhich and how many samples were used to characterize the pre‐event water composition. Analyses based ona limited number of groundwater samples likely underestimate the real uncertainty and can give a falseimpression of accuracy. Our results highlight the importance of representing the variability in the pre‐eventwater composition when applying hydrograph separation analyses. We therefore recommend samplingpre‐event water at multiple locations or estimating the variability based on literature values.