Many management agencies seek to evaluate temporal changes in aquatic assemblages at monitoring sites, but few have sites with ecological time series that are long enough for this purpose. Trends in aquatic-invertebrate and fish assemblage composition were assessed at 27 long-term monitoring sites in the north-central and northeastern United States. Temporal changes were identified using serial trend analysis. Sites with significant serial trends were further evaluated by relating explanatory environmental variables (e.g., streamflow, habitat, and water chemistry) to changes in assemblage composition. Significant trends were found at 19 of 27 study sites; however, differences in the sensitivity of the aquatic fauna to environmental stressors were identified. For example, significant trends in fish assemblages were found at more sites (15 of 27) than for aquatic-invertebrate assemblages (10 of 27 sites). In addition, trends in the invertebrate assemblage were most often explained by changes in streamflow processes (e.g., duration and magnitude of low- and high-flows, streamflow variability, and annual rates of change), whereas trends in the fish assemblage were more related to changes in water chemistry. Results illustrate the value of long-term monitoring for the purpose of assessing temporal trends in aquatic assemblages. The ability to detect trends in assemblage composition and to attribute these changes to environmental factors is necessary to understand mechanistic pathways and to further our understanding of how incremental anthropogenic alterations modify aquatic assemblages over time. Finally, this study's approach to trends analysis can be used to better inform the design of monitoring programs as well as support the ongoing management needs of stakeholders, water-resource agencies, and policy makers.