Site-specific temporal trends in algae, benthic invertebrate, and fish assemblages were investigated in 15 streams and rivers draining basins of varying land use in the south-central United States from 1993–2007. A multivariate approach was used to identify sites with statistically significant trends in aquatic assemblages which were then tested for correlations with assemblage metrics and abiotic environmental variables (climate, water quality, streamflow, and physical habitat). Significant temporal trends in one or more of the aquatic assemblages were identified at more than half (eight of 15) of the streams in the study. Assemblage metrics and abiotic environmental variables found to be significantly correlated with aquatic assemblages differed between land use categories. For example, algal assemblages at undeveloped sites were associated with physical habitat, while algal assemblages at more anthropogenically altered sites (agricultural and urban) were associated with nutrient and streamflow metrics. In urban stream sites results indicate that streamflow metrics may act as important controls on water quality conditions, as represented by aquatic assemblage metrics. The site-specific identification of biotic trends and abiotic–biotic relations presented here will provide valuable information that can inform interpretation of continued monitoring data and the design of future studies. In addition, the subsets of abiotic variables identified as potentially important drivers of change in aquatic assemblages provide policy makers and resource managers with information that will assist in the design and implementation of monitoring programs aimed at the protection of aquatic resources.