Habitat use by fishes in groundwater-dependent ecosystems with springs and spring-fed creeks is not widely studied or well understood. We evaluated habitat use by three disjunct populations of fish species (Phoxinus erythrogaster, Nocomis asper and Etheostoma microperca) and, a widespread species, E. spectabile in spring-fed streams draining the Arbuckle-Simpson aquifer of southern Oklahoma. Habitat preference for each species was classified based on depth, velocity, substrate and cover. Phoxinus erthyrogaster and N. asper were associated with pools with little cover, while E. microperca was found in heavily vegetated areas. Etheostoma spectabile used habitat in riffles with rapid velocity and large substrate types. We classified habitat selection and avoidance with Chesson's α and observed significant differences in habitat use among species in the study sites. Overall differences in habitat use for P. erythrogaster among the three study sites were primarily related to differences in available habitat between springs. Our study provides vital ecological information about disjunct populations of groundwater-dependent fishes in an aquifer that is experiencing development pressure for water abstraction.