Although well-known interactions among river hydrological and thermal regimes have been described, both have yet to be analysed together for assessing guild responses across rivers, including those subjected to river regulation. We compared between-guild responses to varying hydrological and thermal regimes across 25 unregulated and regulated rivers in Canada, by testing the relationship between dominant patterns of flow and thermal variation and fish density and biomass among different types of traits used to describe guilds. Fish guild models were significantly explained (R²_Adj= 25- 44%) and predicted (R²_CV= 35- 76%) by explanatory variables representing river flow and thermal regimes across rivers, whereas total fish density and biomass were not. The best explanatory models were found for traits describing habitat and trophic guilds (R²_Adj = 0.44 and R²_Adj = 0.41, respectively), suggesting that habitat and trophic traits are more closely associated with variables depicting river flow and thermal variability than other guild traits. Specifically, flow and thermal variables depicting the magnitude of summer water temperatures and intra-annual flow variability were consistently selected for as independent drivers of fish guild models (> 86% of models), pointing to the importance of integrating thermal regimes in hydro-ecological studies. Our model results allowed us to explore the functional relationships that may cause specific fish assemblages to be more vulnerable to variations in flow and thermal regimes, especially those arising from river regulation. For example, the guild that preferred cold water, riffle-type habitats, and was intolerant to environmental perturbations was positively related with large variability of annual flows and negatively associated with the magnitude of summer temperatures. Likewise, habitat guilds that preferred warm water temperatures were associated with regulated rivers.These findings identified the different guild trait-environment relationships across rivers and highlighted the importance of more comprehensive flow and thermal regime conservation for which management efforts focused on maintaining the ecological integrity of rivers should rely on.