Stream drying and in-stream barriers can influence migration and recolonization, yet we know little about how these impact population genetics of fishes. We analyzed microsatellite DNA from two common headwater fishes, Longear Sunfish ( Lepomis megalotis ) and Highland Stoneroller (Campostoma spadiceum), to compare population genetics of fishes in streams with varying degrees of connectivity. We sampled perennial and intermittent streams including examples with and without anthropogenic structures (road crossings). An additional perennial stream with a large waterfall was added to examine potential effects of a natural structure. We collected between 24 and 30 individuals of each species from three equidistant locations along each of five study streams. We found that Longear Sunfish and Highland Stoneroller populations experienced greater reductions in gene flow along the intermittent streams than along the perennial streams. However, the greatest reduction in gene flow between sub-populations generally coincided with the presence of permanent in-stream structures. Our study highlighted how overall genetic connectivity of fish populations is reduced by stream drying and showed that these effects can be exacerbated by in-stream barriers.