We investigated the relations among 83 hydrologic condition metrics (HCMs) and changes in algal, invertebrate, and fish communities in five metropolitan areas across the continental United States. We used a statistical approach that employed Spearman correlation and regression tree analysis to identify five HCMs that are strongly associated with observed biological variation along a gradient of urbanization. The HCMs related to average flow magnitude, high-flow magnitude, high-flow event frequency, high-flow duration, and rate of change of stream cross-sectional area were most consistently associated with changes in aquatic communities. Although our investigation used an urban gradient design with short hydrologic periods of record (≤1 year) of hourly cross-sectional area time series, these five HCMs were consistent with previous investigations using long-term daily-flow records. The ecological sampling day often was included in the hydrologic period. Regression tree models explained up to 73, 92, and 79% of variance for specific algal, invertebrate, and fish community metrics, respectively. National models generally were not as statistically significant as models for individual metropolitan areas. High-flow event frequency, a hydrologic metric found to be transferable across stream type and useful for classifying habitat by previous research, was found to be the most ecologically relevant HCM; transformation by precipitation increased national-scale applicability. We also investigated the relation between measures of stream flashiness and land-cover indicators of urbanization and found that land-cover characteristic and pattern variables, such as road density, percent wetland, and proximity of developed land, were strongly related to HCMs at both a metropolitan and national scale and, therefore, may be effective land-use management options in addition to wholesale impervious-area reduction.