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Karst spring systems provide permanent water sources in semi-arid to arid environments, supporting a diverse aquatic community. Numerous karst streams in the Edwards Plateau and Trans-Pecos regions of Texas support two distinct fish assemblages, spring-associated species and riverine species. Previous studies have identified constant water temperature as the segregating factor but have lacked the ability to effectively distinguish temperature from headwater influence. Multiple spring sources along a 64-km course of a semi-arid stream allowed an opportunity to assess the influence of temperature and other environmental variables on spatiotemporal patterns in the fish assemblage. Physical parameters (10.6%), site (4.9%), and season (1.6%) explained significant variation within the Devils River fish assemblage. Current velocity, depth, and vegetation were among the strongest factors explaining fish segregation. Water temperature influenced seasonal movement of spring-associated species into spring outflow habitats during summer and winter and resulted in a segregated assemblage. This study suggests spring flow (i.e., current velocity and depth) and constant water temperature are leading segregating factors and important for maintaining parapatric assemblages. Published by Elsevier Ltd.