Using the mitochondrial cytochrome oxidase I (COI) gene, we assessed the phylogeographic structure of Prosimulium neomacropyga, a black fly (Simuliidae) whose distribution in the US Southern Rockies ecoregion is limited to alpine tundra streams. Given high habitat specificity, lack of hydrological connection between streams, and a terrestrial environment restrictive to insect flight, we hypothesized limited gene flow. A spatially nested sampling design showed that grouping populations according to high-elevation ‘islands’ of alpine tundra (which typically include headwater streams of > 1 watershed) explained a significant proportion of genetic variation while grouping streams according to major watershed (across islands) did not. Nested clade analysis and isolation-by-distance (IBD) relationships further implicated limited ongoing gene flow within but not among the isolated alpine islands. IBD was strong among five streams within an individual island using each of four alternative models of pairwise landscape connectivity for flying insects. Results of all landscape models were positively correlated, suggesting that straight-line distance is an acceptable surrogate for presumably more biologically meaningful connectivity measures in this system. IBD was significantly weaker across the entire study area, comprised of three separate islands. Overall, population structure was significant with FST = 0.38, suggesting limited dispersal across a small spatial extent.