The National Estuarine Research Reserve System represents a diverse collection of ecosystems among which environmental conditions differ dramatically, making it inherently difficult to determine the extent to which patterns and properties identified in one estuary are transferable to those of any other. The primary objective of our study was to develop a multivariate classification framework for comparison of these estuaries and identify the primary sources of environmental variability in each. Using a 4-year dataset from the National Estuarine Research Reserve System-Wide Monitoring Program, combined with principal components analysis, we identified distinct patterns among 21 reserves that allowed grouping based on the primary factors shaping physicochemical variability. Salinity and temperature were the primary factors shaping variability in the reserves, an observation that was corroborated by similar multivariate analysis of data from 33 published studies of non–National Estuarine Research Reserve systems representing a wide range of coastal and estuarine waters. We then investigated the effect of temperature and salinity on biological processes in these systems by using the ratio of bacterial production to chlorophyll-a as a response variable. Salinity and temperature had different but significant effects on bacterial production/chlorophyll-a ratios, suggesting in turn that these properties contribute to the balance between autotrophic and heterotrophic planktonic processes in estuarine ecosystems. Our study confirms the universal role of salinity and temperature in shaping the variability among even the most diverse systems and provides a valuable classification framework for comparison of reserves within the context of the entire National Estuarine Research Reserve System. Use of this classification approach may provide insight into the extent to which results from investigative studies in one reserve may be applicable to others, a valuable application when the effect of environmental stressors is considered.