Responses of ecosystems to human-induced stress vary in space and time, because both stressors and ecosystem vulnerabilities vary in space and time. Presently, ecosystem impact assessments mainly take into account variation in stressors, without considering variation in ecosystem vulnerability. We developed a method to address ecosystem vulnerability variation by quantifying ecosystem vulnerability distributions (EVDs) based on monitoring data of local species compositions and environmental conditions. The method incorporates spatial variation of both abiotic and biotic variables to quantify variation in responses among species and ecosystems. We show that EVDs can be derived based on a selection of locations, existing monitoring data and a selected impact boundary, and can be used in stressor identification and ranking for a region. A case study on Ohio's freshwater ecosystems, with freshwater fish as target species group, showed that physical habitat impairment and nutrient loads ranked highest as current stressors, with species losses higher than 5% for at least 6% of the locations. EVDs complement existing approaches of stressor assessment and management, which typically account only for variability in stressors, by accounting for variation in the vulnerability of the responding ecosystems.