We used mitochondrial cytochrome c oxidase subunit I to infer the demographic history of Acroneuria frisoni Stark & Brown, a widespread eastern North American stonefly. We sequenced 1510 base pairs from 348 individuals from 36 populations, obtained 160 haplotypes, and tested 3 major hypotheses: 1) Wisconsinan and Illinoian glaciations were vicariant events that separated western and eastern populations; 2) the Mississippi River floodplain was a postglacial barrier to dispersal of eastern and western populations; and 3) at least 2 genetically distinct glacial refugia existed, east and west of the Mississippi River. We also investigated other barriers, pathways to dispersal, and the relative importance of refugia to repopulation of northern areas. Species-distribution modeling of its Wisconsinan-glacial-age distribution placed A. frisoni at the far southern end of the current distribution and in other areas further south and west than it is currently known, and separated western and eastern populations. Eastern and western populations accounted for 70% of the variation in the data set (analysis of molecular variance), and the Mississippi River was a major barrier to gene flow (Bayesian phylogenetic analysis). We identified a western, Ozark Mountains clade (OZK), a Midwest clade (MDW) in central and western Tennessee, and an Appalachian Mountains clade (APP) in Tennessee and Pennsylvania. Members of the MDW clade recolonized once-glaciated and glacially influenced areas north of the Ohio River. Members of the APP clade appear to be confined to the Tennessee River valley and the Ridge and Valley province of the Appalachian Mountains. The Mississippi River floodplain prevented members of the OZK clade from recolonizing northern areas, except in southern Illinois. Comparison to other species with similar distributions points to refuge use and recolonization similar to that of a megalopteran. Our data will guide reintroduction of A. frisoni into areas of the Midwest from which it has been extirpated.