We describe a new species of Nothonotus from the Caney Fork River drainage, Tennessee, USA, and assess morphological variation in N. microlepidus and N. sanguifluus with specimens sampled from all known populations. We used Linear Discriminant Analysis (LDA) on a meristic dataset from 831 individuals to delimit species of the N. maculatus species group endemic to the Cumberland River drainage. We assigned populations to groups in the LDA on the basis of phylogenetic lineages identified in previous studies of Nothonotus darters using mitochondrial and nuclear gene DNA sequences. The discriminant functions were able to reasonably differentiate among N. microlepidus, N. sanguifluus and the new species of Nothonotus. The discriminant functions did not reliably differentiate among N. sanguifluus populations sampled from different tributaries in the Cumberland River drainage. Two populations of N. sanguifluus were not included in the LDA because of limited material and thus could not be assigned to a group; these are tentatively assigned to N. sanguifluus partially on the basis of predictions using the model from the LDA and the estimated phylogeny. Phylogenetic and multivariate analyses of morphological characters allow differentiation of the new species from its closest relatives, N. microlepidus and N. sanguifluus. Additionally, the new species can be differentiated from other species in the N. maculatus species group with a combination of characters, including having red in all median fins of breeding-condition males, the presence of a suborbital bar, extensive red in the pectoral fins, and lower counts of lateral line scales as compared with N. sanguifluus. With the description of this new species of Nothonotus there are now five described endemic darters occurring in the Caney Fork above Great Falls.