The current literature treats the plains killifish as a single species (Fundulus zebrinus). However, a recent range-wide survey of genetic variation detected two distinct groups. The presence of the two groups suggests that the plains killifish may actually represent two species (F. zebrinus and F. kansae), as has been proposed in prior taxonomic treatments. Previous molecular studies within the genus Fundulus have relied upon the mitochondrial cytochrome b gene. I generated a set of cytochrome b sequences in order to compare the phylogenetic support for the putative species F. zebrinus and F. kansae with that found in other accepted sister species of Fundulus. Both neighbor-joining and maximum parsimony analyses recovered two clades of plains killifish that were strongly supported by a variety of measures including branch lengths, decay indices and bootstrap proportions. The strength of support for the putative F. kansae and F. zebrinus clades was comparable to, or better than, similar measures for six other sister species pairs of Fundulus. While the molecular data supported the recognition of two species of plains killifish, the groups recognized were not in agreement with the range descriptions previously proposed for the two species.