Altitudinal gradients in Sub-Antarctic freshwater systems present unique opportunities to study the effect of distinct environmental gradients on benthic macroinvertebrate community composition and dispersal. This study investigates patterns in biodiversity, dispersal and population genetic structure of benthic macroinvertebrate fauna across an altitudinal gradient between two watersheds on Navarino Island in southern Chile. Patterns in diversity, density, evenness and functional feeding groups were not significantly different across the altitudinal gradient in both the Windhond and Róbalo Rivers. Taxa richness in both rivers generally increased from the headwaters of the river to the mouth, and functional feeding group patterns were consistent with the predictions of the River Continuum Concept. Population genetic structure and gene flow was investigated by sampling the mitochondrial cytochrome oxidase I gene in two invertebrate species with different dispersal strategies. Hyalella simplex (Amphipoda) is an obligate aquatic species, and Meridialaris chiloeense (Ephemeroptera) is an aquatic larvae and a terrestrial winged adult. Contrasting patterns of population genetic structure were observed. Results for Hyalella simplex indicate significant differentiation in genetic structure in the Amphipod populations between watersheds and lower genetic diversity in the Róbalo River samples, which may be a result of instream dispersal barriers. Meridialaris chiloeense exhibited weak population structure but higher genetic diversity, which suggests this species is able to disperse widely as a winged adult.