The Central Highlands of North America have an old and complex geological and biological history, with extensive unexplored cryptic diversity. We examined the species limits of the Fundulus catenatus species group (F. catenatus, Fundulus bifax, and Fundulus stellifer) using two phylogenetic approaches. Fundulus catenatus was densely sampled from five geographically disjunct regions across the eastern USA. We sequenced four nuclear introns and used species tree analyses and two species delimitation methods to determine historical relationships and investigate species diversity within the group. Both methods strongly supported the presence of two reciprocally monophyletic species within F. catenatus. Species tree analysis of four nuclear introns (stx5a, ncl1, rpsa, and rps3) from the F. catenatus species group (including a tip for F. sp. cf. catenatus) recovered the two Mobile Basin species as sister to a strongly supported clade of F. catenatus and F. sp. cf. catenatus. Cytochrome b sequences were used in phylogeographical analyses of the two putative species. In one species we identified signatures of population expansion whereas the other exhibited genetic structure consistent with isolation of populations.