The phylogenetic relationships within the Centrarchid genus Enneacanthus and the role of regional historical biogeography in their radiation was examined. Based on complete mitochondrial (mt) control region sequences, E. chaetodon is a monophyletic taxon that is sister to an ‘E. gloriosus E. obesus’ clade. However, the current data indicate that E. gloriosus and E. obesus are not monophyletic taxa and appear to demonstrate either incomplete lineage sorting or a polyphyletic E. obesus. Based on molecular clock divergence estimates, the hypothesis of Pleistocene glacial cycles promoting speciation was not supported for either E. chaetodon or E. gloriosus, but is supported in having been a factor in the origins of E. obesus. Even though not a common force in terms of speciation, the Pleistocene oscillations do appear to have promoted subsequent diversification and dispersal (range expansion) for all three of these species.