Investigations of phylogenetic relationships using comparative morphological characters have led to the hypothesis that Ammocrypta is the sister taxon of the Etheostoma subgenus Ioa. Subsequent molecular analyses have not recovered this proposed relationship. In this investigation, variation in complete cytochrome b sequences sampled from 27 percid species, representing all darter genera and all species of Ammocrypta, is examined in a phylogenetic context. Third codon purine transitions are saturated when all darter species examined are compared; however, saturation is not detected in any character class among species of Ammocrypta. Maximum-parsimony analysis, which excludes potentially homoplasious character classes, and maximum-likelihood analysis of cytochrome b sequences statistically reject the hypothesis that Ammocrypta is the sister taxon of the Etheostoma subgenus Ioa. Etheostoma is not recovered as monophyletic; however, the hypothesis of monophyly cannot be rejected using maximum-parsimony and maximum-likelihood methods. Morphological characters are combined with cytochrome b sequences in a total evidence analysis of relationships within Ammocrypta. A novel hypothesis of relationships is proposed, which includes a sister-taxon relationship between A. clara and A. pellucida. With regard to relationships among species of Ammocrypta, the morphological data do not significantly conflict with the cytochrome b data. Morphological characters provide additional synapomorphies supporting the hypothesis that A. beani and A. bifascia are sister taxa. The biogeographic implications of the total evidence analysis are discussed, and the continued taxonomic placement of Ammocrypta in Etheostoma is discouraged.