Madtom catfishes of the genus Noturus are a well-known component of the North American ichthyofauna. Original nucleotide sequence data were collected from mitochondrial (cytochrome b) and nuclear (recombination activating gene 2) genes and used to estimate genetic variation and infer phylogenetic relationships among and within species of Noturus. Mitochondrial sequences were variable among species and several species were found to contain considerable genetic diversity. Relationships among members of the subgenus Rabida were resolved and in many cases well supported. Relationships among members of the subgenus Schilbeodes were poorly resolved. Previous phylogenetic hypotheses and the traditional classification (except the furiosus species group) were rejected in their explicit form according to the Kishino-Hasegawa and Shimodaira-Hasegawa tests of tree score difference.