Brook trout (Salvelinus fontinalis) reside in a range of habitats including headwater streams where populations may become periodically isolated. Demographic consequences of population isolation include increased potential for localized extinction. Genetic consequences for population isolation, or populations of sustained small numbers, include increasing susceptibility to inbreeding, the expression of negative fitness traits, and overall loss of genetic variability, which may increase the risk of localized extinction. Reestablishment of connectivity -with larger populations through periodic gene flow into these small populations may act to offset loss of genetic diversity. Restoring population connectivity and degraded headwater habitats, especially those that historically harbored wild brook trout populations has been a focus of restoration efforts. To understand the degree of genetic isolation among headwater brook trout populations, we examined how variability in connectivity between brook trout populations bas resulted in the partitioning of genetic variation within a number of headwater brook trout populations in two locations: Nash Stream, New Hampshire, and Pennsylvania. Both locations bad multiple headwater brook trout populations, but varied by how populations were potentially isolated. In Nash Stream, potential causes of isolation were culverts or waterfalls. In Pennsylvania, isolation could be due to larger geographic distance, culverts or dams, or areas of poor stream quality. In both locations, overall estimates of variation in allele frequency among populations were high, indicating that populations were significantly genetically different, but the effect of the different barrier types varied based on the likely amount of gene flow allowed. For example, in Pennsylvania, significant correlations of genetic diversity were only observed with the presence of impaired stream sections, but not geographic distance or barrier effect. Understanding the effects of barriers on genetic diversity and gene flow can be useful to guide efforts to restore connectivity between populations.