Catfishes of the genus Ameiurus are a well-known component of the North American ichthyofauna. J. G. Lundberg estimated phylogenetic relationships among species of Ameiurus according to morphological variation. Two clades containing the extant species were recovered: the natalis clade (Ameiurus natalis, Ameiurus melas, and Ameiurus nebulosus) and the catus clade (Ameiurus serracanthus, Ameiurus catus, Ameiurus platycephalus, and Ameiurus brunneus). Original nucleotide sequence data were collected from mitochondrial (cytochrome b) and nuclear (recombination activation gene 2) genes and used to test the predictions of the hypothesis based on morphological data. The monophyly of both natalis and catus clades was rejected by the nucleotide dataset and an alternative phylogenetic hypothesis is provided.