Phylogenetic relationships among species of Percina are unresolved. Previous systematic studies of Percina have resulted in the recognition of nine subgenera, diagnosed by external morphological characters. Throughout the history of darter taxonomy characters such as large body size, high meristics, drab coloration, and exploitation of a hyperbenthic habitat have been interpreted as pleisiomorphic. Most species of Percina exhibit these characters, and have been hypothesized to represent the “primitive” lineage of darters. The hypotheses that each of the polytypic subgenera of Percina are monophyletic and that the previously defined primitive characters are pleisiomorphic, have not been investigated with cladistic analyses. In this investigation, complete gene sequences of the mitochondrially encoded cytochrome b were collected from a total of 79 individual specimens, representing nine of 10 percid genera and all 40 species of Percina. Observed patterns of cytochrome b evolution were very similar to those previously reported in other percid fishes. Maximum-parsimony and maximum-likelihood analyses were generally congruent. The majority of subgenera (Percina, Imostoma, Cottogaster, Swainia, and Odontopholis) were recovered as monophyletic in most analyses. The subgenera Alvordius, Hadropterus, and Ericosma were never recovered as monophyletic; however, monophyly of Hadropterus and Ericosma could not be rejected in statistical analyses of maximum-likelihood score differences. As a result of these phylogenetic analyses, a novel classification of Percina species is proposed. The use of subgenera in Percina taxonomy is abandoned in favor of the recognition of monophyletic “species clades.” Reconstruction of character evolution on the hypothesized phylogenetic relationships suggest that previously identified pleisiomorphic character states in darters may actually be derived within Percina. Hypothesis testing of derived and ancestral traits in darters is complicated by uncertainty in ancestral character state reconstruction. Contributing to the lack of confidence in character optimization are inadequate sampling of Etheostoma species, short internal branches on the phylogeny, and a high frequency of character change across the entire diversity of darters.