We examined the genetic structure and phylogeography of populations of the stonefly Peltoperla tarteri in the Southern Appalachians to determine the extent and likely mechanism for dispersal of this stream insect. A 454-base-pair (bp) portion of the mitochondrial control region was sequenced from a minimum of 20 individuals from eight populations. Pairwise FST and exact tests showed high levels of differentiation among almost all populations except those on the same stream. amova analysis detected significant genetic differentiation between streams within drainages (φSD = 0.14, P < 0.001), and there was a slight positive correlation between aquatic distance and genetic distance (r = 0.295, P = 0.03). According to nested clade analysis, the present day pattern of genetic variation in P. tarteri is the result of a historical range expansion coupled with restricted gene flow with isolation by distance. Together, these analyses suggest that adult dispersal is limited and that movement by larvae is the primary dispersal mechanism for P. tarteri.