The Mississippi River Basin supports the richest fish fauna in eastern North America and has played a key role in the maintenance of fish biodiversity, especially as a refuge for freshwater fishes during glaciations. In this study, we investigated the phylogeography of the bluegill sunfish, Lepomis macrochirus, in eastern North America, using complete sequence of the mitochondrial ND1 gene from 369 samples collected at 15 sites. Phylogenetic analysis revealed two major lineages (northern and southern clades) in a parsimony network. A sympatric distribution of the lineages was widely observed in the Mississippi Basin. Sequence diversity in the two lineages was significantly lower in glaciated regions around the Great Lakes than in unglaciated regions. The two lineages were estimated to have diverged in the Kansan glaciation, and refugia for both existed around the Ouachita Highlands. The southern clade dispersed during the Yasmouth Interglacial, prior to the dispersal of the northern clade during the Sangamon Interglacial. In the northern clade, low genetic diversity and population fragmentation inferred by nested clade analysis (NCA) were considered due to bottleneck events in the Wisconsin glaciation, while the southern clade showed isolation by distance in a Mantel test. A difference in demographic fluctuation suggests that sympatry of the two lineages has resulted from recent secondary admixture through the range expansion of the northern clade in the post-Pleistocene. Large-scale admixture of multiple mtDNA lineages in L. macrochirus, which has not been recorded in other fishes in the Mississippi River Basin, may result from their high vagility.