Understanding dispersal and gene flow patterns of species is fundamental to the success of conservation strategies.  For small, benthic, freshwater fishes there are few studies of intraspecific gene flow and population structure.  The biology and life history of these fishes indicate that many are probably dispersal limited.  The Fantail Darter is one example.  Intrinsic and extrinsic features of this species predict that gene flow among populations may be limited and that significant genetic structure may occur at small spatial scales.  To test these predictions, we examined genetic variation in the mitochondrial ND2 gene for 200 individuals from 41 localities from the Pee Dee, Santee, Savannah and Cape Fear River drainages (Atlantic Slope), and the upper New and Tennessee River systems (Ohio River).  Examination of haplotype divergences, relationships and geographic distributions revealed high levels of population subdivision across most drainage divides and at smaller spatial scales within drainages. Haplotypes were structured primarily by collection site with few instances of shared haplotypes among collection sites. Preliminary evidence suggests that within drainages, distances of seven river kilometers may be sufficient to prevent gene flow among populations of this species. These findings have important conservation implications for other benthic fishes.