The alteration of paleodrainage river connections has shaped patterns of speciation, genetic diversity, and the geographic distribution of the species-rich freshwater fauna of North America. The integration of ancestral range reconstruction methods and divergence time estimates provides an opportunity to infer paleodrainage connectivity and test alternative paleodrainage hypotheses. Members of the Orangethroat Darter clade, Ceasia, are endemic to southeastern North America and occur north and south of the Pleistocene glacial front, a distributional pattern that makes this clade of closely related species an ideal system to investigate the number and location of glacial refugia and compare alternative hypotheses regarding the proposed evolution of the Teays-Mahomet paleodrainage. This study utilized time-calibrated mitochondrial and nuclear gene phylogenies and present-day geographic distributions to investigate hypothesized Teays-Mahomet River connections through time using a dispersal-extinction cladogenesis framework (DEC). Results of DEC ancestral area reconstructions indicate that the Teays-Mahomet River was a key dispersal route between disjunct highland regions connecting the Mississippi River tributaries to the Old-Ohio Drainage minimally at two separate occasions during the Pleistocene. There was a dynamic interplay between paleodrainage connections through time and postglacial range expansion from three glacial refugia that shaped the current genetic structure and geographic distributions of the species that comprise Ceasia.