ABSTRACT Two new ctenacanthiform sharks representing two families, Ctenacanthidae and Heslerodidae, have been identified from the Middle to Late Mississippian marine sediments from Mammoth Cave National Park, Kentucky, and two Late Mississippian marine horizons in northern Alabama. The ctenacanthid, Troglocladodus trimblei, gen. et sp. nov., is known from isolated teeth from the Middle Mississippian St. Louis Formation and Ste. Genevieve Formation of Mammoth Cave and the Late Mississippian Bangor Limestone of northern Alabama. Troglocladodus gen. nov. is characterized by broad median cusps, pronounced longitudinal cristae, multiple intermediate cusps, and labiolingually shortened tooth bases. The heslerodid, Glikmanius careforum sp. nov., is known from isolated teeth and visceral arches from the Middle Mississippian St. Louis Formation and Ste. Genevieve Formation and the Late Mississippian Haney Formation, a Hartselle Sandstone-equivalent shale interval, and Bangor Limestone. Glikmanius careforum sp. nov. has proportionately distinct teeth among species of Glikmanius, with more robust and shorter cusps. The palatoquadrate of G. careforum has a short palatine ramus, otic process that is dorsoventrally deep and less expanded antero-posteriorly similar to Heslerodus and Dracopristis, and an elongated quadrate process like Heslerodus. The Meckel’s cartilage is less dorsoventrally deep than Dracopristis. These two new ctenacanth taxa add important information on the diversity of Ctenacanthiformes suggesting three major lineages within the order. Ctenacanths have a rich fossil history from the Late Devonian to the Middle Permian evolving a variety of tooth types and small to large body sizes.