Ecological classifications of stream ecosystems have been used to develop monitoring programs, identify reference and impacted systems, and focus conservation efforts. One of the most influential, but highly variable, components of stream ecosystems is water temperature but few geographically broad-scale and long-term programs exist to assess and monitor temperatures. This study evaluated if existing ecological classifications could be used to categorize the similarities and differences in stream temperatures across the Ontario portion of the Great Lakes Basin. Concordance between the spatial variability in temperatures and an existing ecological classification would support the use of that classification to define areas with similar temperatures, guide the development of a monitoring program, and inform management programs. The five classifications evaluated were the ecoregions and ecodistricts defined in the National Ecological Framework for Canada, the ecoregions and ecodistricts defined in the Ecological Land Classification of Ontario, and the aquatic ecosystem units defined in the Aquatic Ecosystem Classification (AEC) for the Ontario portion of the Great Lakes Basin. Hierarchical linear modelling and corrected Akaike Information Criterion indicated that the ecodistrict classifications characterized more of the spatial variability in temperatures than the ecoregion and AEC classification but temperatures were more variable among sites within classes than between classes. Therefore, none of the existing ecological classifications could be used to characterize thermal variability. Future research should examine if the inability of the existing classifications to capture the thermal variability translates into inaccurate classification of other ecosystem components such as water quality, and macroinvertebrate and fish assemblages.