The cumulative impacts of urban land use on stream flow regimes and lotic ecosystems are poorly understood. Moreover, flow assessments using daily or monthly flows cannot adequately characterize event-scale flow dynamics in urbanizing watersheds. Accordingly, this empirical research examined high temporal resolution (15-minute) growing season hydrologic records in the Greater Toronto Region, Canada. Hydrologic records were matched with rainfall records to include precipitation in models. The first phase of research identified temporal trends in total runoff, rising limb event flows and rising limb accelerations in two watersheds. Results indicated dramatic changes: over a 42-year period, total seasonal discharge increased 45% in the Don and Humber Rivers during a period of stable rainfall patterns. Peak event flows and event flow variability also increased temporally. The second phase of research comprised a spatial analysis of twenty-seven watersheds ranging from 38 km2 to 806 km2 undertaken along an urban land use gradient from less than 0.1% to 88%. Urban land use had a very strong influence on total runoff and event scale runoff. Changes in runoff characteristics began at urban cover under 4%. Event flow acceleration increased, causing maximum runoff to be reached sooner as urban cover increased. The total runoff model had an interaction between watershed size and urban land use. Smaller watersheds are disproportionately affected by urbanization, as also demonstrated by the fractal relationship of stream network length and watershed area. The third phase identified associations of fish species richness with event-scale hydrologic characteristics in eight watersheds using fish data spanning approximately five decades. Maximum event flow acceleration and skew in instantaneous runoff explained a higher proportion of variation than urban percent in empirical models. Historic fish data are difficult to obtain and pose analytical challenges. By using high temporal resolution flow data, the research provides a new understanding of changes in event-scale flow regime dynamics associated with urbanization and, in turn, association of event-scale hydrology with fish richness. Compromised hydrologic stationarity is attributable to urbanization.