Abstract A new species related to Percina macrocephala is described from the upper Tennessee River drainage. Percina williamsi is distinguished from all species of Percina except P. macrocephala by the presence of a sickle-shaped suborbital bar and a black bar subtending a medial black spot on the caudal-fin base. It is separated from P. macrocephala by having larger scales, including usually 24–26 scales around the caudal peduncle (vs. 27–31 in P. macrocephala), 21–23 transverse scales (vs. 23–26), and 70–77 lateral scales (vs. 76–86). Values for the combination of caudal peduncle, transverse, and lateral scales are usually 120–129 in P. williamsi and 128–141 in P. macrocephala. Phylogenetic analyses of complete mtDNA cytochrome b gene sequences resolve P. macrocephala and P. williamsi as sister species, and intraspecific sampling indicates that mtDNA haplotypes of both species are reciprocally monophyletic. Estimates of divergence times indicate that P. macrocephala and P. williamsi shared a most recent...