The North American cyprinid genus Pteronotropis contains five species distributed in streams and swamps of the Atlantic and Gulf coastal plain. The genus is currently recognized even though previous explicitly phylogenetic analyses have not supported the monophyly of this group. Of particular interest is the relationship between Pteronotropis welaka and Pteronotropis hubbsi, both of which are nest associates with centrarchids. Although these species are morphologically similar, there is limited support for a sister-group relationship between them. We performed phylogenetic analyses of all five species currently recognized in Pteronotropis and five outgroup taxa based on nucleotide sequences of the mitochondrial cytochrome b gene using both parsimony and maximum-likelihood analyses. Pteronotropis hypselopterus and Pteronotropis euryzonus were consistently resolved as sister taxa, as were, P. welaka and P. hubbsi. Pteronotropis signipinnis was sister to the P. welaka plus P. hubbsi clade in likelihood and parsimony analyses when rate variation at third codon positions was taken into account. However, none of the analyses resolved a monophyletic group inclusive of all five species. If Pteronotropis is actually a monophyletic group, our inability to resolve it as such may be a result of sampling of outgroup taxa or the rate of evolution in cytochrome b in these taxa. Additional sampling of taxa, particularly within the genus Notropis is needed to further resolve phylogenetic relationships within Pteronotropis and the relationships of the species to other North American cyprinid taxa. The sister-group relationship between P. welaka and P. hubbsi indicates a single origin of nest association rather than a dual origin as indicated by previous studies.