Environmental and handling stressors are common to every aquaculture system and affect various cultured fish species differently. Understanding these diverse stress responses is crucial for optimizing aquaculture practices. This study investigates the physiological responses to stressors of two fish species representative of cultured species groups in North America. The species selected, channel catfish (Ictalurus punctatus) and alligator gar (Atractosteus spatula), were hypothesized to differ greatly in magnitude of responses. We exposed both species to environmental and handling stressors and evaluated their physiological stress responses using a variety of methods, including analyses of blood and tissue metabolites and measurements of the classical stress hormone cortisol. Additionally, we compared the skin mucus peptide profiles of both species following stress exposure. The results will provide a baseline for understanding physiological responses to stress in these species, be useful for comparing a range of stress levels and the techniques used to measure stress, and offer insights that can inform and improve culture practices in aquaculture.