Headwater-resident fishes may be prone to a high rate of isolation and a pronounced hierarchical genetic structure due to a combination of niche preference and dendritic effects of river networks. Genetic isolation patterns were compared using microsatellites in six headwater fishes, Fundulus olivaceus, Semotilus atromaculatus, Erimyzon claviformis, Etheostoma artesiae, Etheostoma whipplei and Etheostoma parvipinne, in three geographic regions that included drainages of small, medium and large sizes in the southern United States. All species showed hierarchical nesting of genetic populations and there were clear and mostly consistent differences between species and regions that were identified through summary statistics derived from two independent analyses. For species comparisons, a high isolation grouping (increased number of isolated genetic clusters or sites within regions) and a low-isolation grouping (decreased number of clusters or sites) were identified. Species group placement was related to niche breadth along the river continuum and presumed abundance and variability of preferred microhabitats, with increased headwater specialization among species being associated with placement in the high-isolation grouping. There was a weakly significant positive effect of drainage size on the number of isolated clusters or sites across all species. Regional patterns were shared in two species, with the region containing the smallest drainages having lower rates of isolation in both datasets. This study shows the effects of regional and species characteristics on genetic isolation for headwater species, which are especially prone to isolation due to spatial, dendritic effects of river networks.