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Freshwater fish communities segregate along water temperature gradients attributed in part to temperature-mediated physiological processes that affect species fitness. In spring complexes of southwest USA, spring complexes with narrow range of water temperatures are dominated by a community of fishes (i.e., spring-associated fishes), whereas riverine habitats with wide-range of water temperatures are dominated by a different community of fishes (i.e., riverine-associated fishes). The purpose of this study was to test a prediction of the concept that temperature-mediated species performance is a mechanism in maintaining community segregation. We predicted that a spring-associated fish (Largespring Gambusia Gambusia geiseri) would feed first and more often in a pairing with a riverine-associated fish (Western Mosquitofish G. affinis) at an average spring temperature (23 °C) and that the riverine-associated fish would feed first and more often in a pairing with the spring-associated fish at a warm riverine temperature (30 °C). Among four trails consisting of 30 pairings, at the spring complex temperature (23 °C), Largespring Gambusia had a greater number of first feeds (mean ± 1 SD, 5.0 ± 0.82) than Western Mosquitofish (2.5 ± 1.73) and had greater mean number of total feeds (1.9 ± 0.31) than Western Mosquitofish (0.81 ± 0.70). At the riverine environment temperature (30 °C), Western Mosquitofish had a greater number of first feeds (5.25 ± 1.71) than Largespring Gambusia (2.5 ± 1.73) and had greater mean number of total feeds (2.78 ± 1.05) than Largespring Gambusia (0.94 ± 0.68). Our findings suggest that temperature-mediated species performance could be maintaining segregation between the two fish communities. This study benefits our understanding of distributional patterns and improves threat assessments of stenothermal aquatic organisms.