River regulation can alter the frequency and magnitude of subdaily flow variations causing major impacts on ecological structure and function. We developed an approach to quantify subdaily flow variation for multiple sites across a large watershed to assess the potential impacts of different dam operations (flood control, run-of-river hydropower and peaking hydropower) on natural communities. We used hourly flow data over a 9-year period from 30 stream gages throughout the Connecticut River basin to calculate four metrics of subdaily flow variation and to compare sites downstream of dams with unregulated sites. Our objectives were to (1) determine the temporal scale of data needed to characterize subdaily variability; (2) compare the frequency of days with high subdaily flow variation downstream of dams and unregulated sites; (3) analyse the magnitude of subdaily variation at all sites and (4) identify individual sites that had subdaily variation significantly higher than unregulated locations. We found that estimates of flow variability based on daily mean flow data were not sufficient to characterize subdaily flow patterns. Alteration of subdaily flows was evident in the number of days natural ranges of variability were exceeded, rather than in the magnitude of subdaily variation, suggesting that all rivers may exhibit highly variable subdaily flows, but altered rivers exhibit this variability more frequently. Peaking hydropower facilities had the most highly altered subdaily flows; however, we observed significantly altered ranges of subdaily variability downstream of some flood-control and run-of-river hydropower dams. Our analysis can be used to identify situations where dam operating procedures could be modified to reduce the level of hydrologic alteration. ?? 2009 John Wiley & Sons, Ltd.