Structural connectivity and dispersal ability are important constraints on functional connectivity among populations. For aquatic organisms that disperse among stream corridors, the regional structure of a river network can, thus, define the boundaries of gene flow. In this study, we used mitochondrial DNA (mtCO1 barcoding gene) to examine the genetic diversity and population structure of a caddisfly with strong dispersal capabilities, Hydropsyche oslari (Trichoptera:Hydropsychidae), in the topologically-diverse Colorado River Basin. We expected to find less genetic differentiation among populations of H. oslari within the Upper Basin, which has a dense dendritic network of perennial tributaries that allow for greater potential dispersal and gene flow, than among populations within the arid and sparse river network of the Lower Basin. We also expected to find genetic differentiation among H. oslari in the Upper and Lower Basins because contemporary populations are geographically distant from each other and have been separated by a >300-km-long reservoir (Lake Powell) for ½ a century. Consistent with these predictions, we found that populations of H. oslari within the Upper Basin had more shared haplotypes and less nucleotide diversity (π = 0.001–0.008) than H. oslari within the Lower Basin (F_ST = 0.01, π = 0.014–0.028). However, populations were genetically more structured in the Upper Basin (F_ST = 0.47) than in the Lower Basin (F_ST = 0.01). We also found that populations in the Upper and Lower Basin are entirely genetically differentiated (S_nn = 1), suggesting that these 2 populations were isolated thousands of years before the 1963 closure of Glen Canyon Dam and subsequent filling of Lake Powell. The most similar haplotypes among the 2 basins represent a 5.4% difference, which indicates the presence of a species complex within H. oslari.