The Kentucky Arrow Darter, Etheostoma spilotum, endemic to the upper Kentucky River basin of eastern Kentucky, is redescribed and recognized as a distinct species closely related to E. sagitta in the upper Cumberland River basin and E. nianguae in the Osage River drainage (Missouri River basin). Originally described as a subspecies of E. nianguae, it was later considered a full species and then a subspecies of E. sagitta, based on close geographic proximity to Cumberland basin populations and overlapping meristic variation interpreted as character intergradation. We present meristic, morphometric, and genetic data that support species-level recognition of E. spilotum. It differs from E. sagitta by lower counts of total and pored lateral scales, lower counts of caudal peduncle scales, fewer second dorsal-fin rays, and fewer pectoral-fin rays. Interspecific divergence of E. spilotum and E. sagitta is further demonstrated through analyses of variation in the mitochondrial nd2 gene and species delimitation using genome-wide double digest restriction-site associated DNA sequencing. Although allopatrically distributed, both species inhabit upland headwater streams on the Cumberland Plateau and have similar life history characteristics. Endemism, fragmented distributions, and low densities and genetic diversity within populations make these species extremely vulnerable to anthropogenic activities. Etheostoma spilotum was federally listed as threatened in 2016 due to degradation of stream habitat and water quality in the upper Kentucky basin that has eliminated the species from a significant portion of its range.