Fish populations may be isolated via natural conditions in geographically separated ecoregions. Although reconnecting these populations is not a management goal, we need to understand how these populations persist across landscapes to develop meaningful conservation actions, particularly for species occupying sensitive karst ecosystems. Our study objective was to determine the physicochemical factors related to the occurrence of four spring-associated fishes. Arbuckle Uplift and Ozark Highlands ecoregions, USA. We used a hierarchical approach to identify habitat relationships at multiple spatial scales. We collected detection data using snorkeling and seining. We examined the physicochemical relationships related to the detection and occurrence of four spring-associated fishes using occupancy modeling in a Bayesian framework. We found physicochemical relationships that differed and were similar between ecoregions for several fishes. For three species, we found different water temperature relationships between ecoregions. Smallmouth bass were ubiquitous in their use of drainage areas in the Ozark Highlands but only associated with the lower network of the Arbuckle Uplift. There were several mirrored relationships between ecoregions, including an interaction between residual pool depth and water temperature, where sites with deeper pools were more likely to be occupied during warmer water temperatures. There were single-species occurrence relationships with percent vegetation and percent agriculture. Lastly, snorkeling was a more efficient sampling method compared to seining for all fishes. Our results indicate stream temperature mitigation may be possible via the maintenance of key channel morphologies, and we identify shared stressors between ecoregions. Channel mitigation to maintain reaches with deeper pools may be an important strategy for maintaining thermal refugia, particularly when considering climate change. Identifying the mechanistic underpinning of other multiscale ecological relationships would be helpful to discern if some of the different ecoregion relationships represent warning signals or interactions with unmeasured biotic or abiotic factors.