Etheostoma xanthovum, the Clarks Darter, is described as a new species endemic to the Clarks River in Kentucky and Tennessee, USA. Etheostoma xanthovum was previously delimited as Etheostoma oophylax, the Guardian Darter, based on morphological trait data. Subsequent to the description of E. oophylax, molecular phylogenetic analyses consistently resolved specimens from the Clarks River system and E. chienense, the Relict Darter, as sister species, which together formed a sister clade to all other sampled populations of E. oophylax. Our analyses of morphological traits, mitochondrial DNA (mtDNA), and genomic sampling using double digest restriction-site associated DNA (ddRAD) sequencing support the distinctiveness of Etheostoma xanthovum. Morphologically, Etheostoma xanthovum, differs from Etheostoma oophylax in the number of dorsal fin rays (12 versus 11), anal fin rays (8 versus 7), and in the number of scale rows around the caudal peduncle (21.8 versus 20.37). Etheostoma xanthovum does not share mtDNA haplotypes with Etheostoma oophylax or Etheostoma chienense. Phylogenomic analysis of an average of 28,448 ddRAD loci per sampled specimen resolves Etheostoma xanthovum and Etheostoma chienense as sister species, and assessment of genomic divergence supports the hypothesis that each of these two species represents a distinct and independently evolving lineage. In addition, we report a range extension of Etheostoma oophylax in the Obion River system, a direct tributary of the Mississippi River.