The Tallgrass Prairie National Preserve (TPNP), Chase County, KS, is located in the Flint Hills, part of the largest remaining tract of tallgrass prairie in the world. The waters of TPNP are inhabited by numerous fish species, but no definitive list of fishes occurring in the preserve has been available previously. We compiled records from published literature and unpublished reports of previous ichthyological sampling and monitoring at TPNP, added information from our own sampling, and present a record of 53 fish species found on the Preserve.