Etheostoma trisella (Trispot Darter), previously thought extirpated in Alabama, was recently rediscovered. Although robust sampling was conducted in the areas surrounding the newly discovered population, more sampling is needed to document previously unknown populations. We took empirical sampling and water samples for eDNA analysis at 30 sites throughout the potential range of Trispot Darter in the Coosa River drainage, AL. We conducted sampling during late winter breeding season, when adults migrate to small headwater streams to spawn, at sites chosen from known breeding sites in St. Clair County through tributaries to Weis Reservoir in Cherokee County. Positive detection occurred at 4/30 (1.3%) sites. Trispot Darter was not detected using empirical sampling.