Developing spatially explicit conservation strategies for stream fishes requires an understanding of the spatial structure of dispersal within stream networks. We explored spatial patterns of stream fish dispersal by evaluating how the size and proximity of connected streams (i.e., stream network topology) explained variation in fish assemblage structure and how this relationship varied with local stream size. We used data from the US Environmental Protection Agency's Environmental Monitoring and Assessment Program in wadeable streams of the Mid-Atlantic Highlands region (n = 308 sites). We quantified stream network topology with a continuous analysis based on the rate of downstream flow accumulation from sites and with a discrete analysis based on the presence of mainstem river confluences (i.e., basin area >250 km²) within 20 fluvial km (fkm) from sites. Continuous variation in stream network topology was related to local species richness within a distance of ∼10 fkm, suggesting an influence of fish dispersal within this spatial grain. This effect was explained largely by catostomid species, cyprinid species, and riverine species, but was not explained by zoogeographic regions, ecoregions, sampling period, or spatial autocorrelation. Sites near mainstem river confluences supported greater species richness and abundance of catostomid, cyprinid, and ictalurid fishes than did sites >20 fkm from such confluences. Assemblages at sites on the smallest streams were not related to stream network topology, consistent with the hypothesis that local stream size regulates the influence of regional dispersal. These results demonstrate that the size and proximity of connected streams influence the spatial distribution of fish and suggest that these influences can be incorporated into the designs of stream bioassessments and reserves to enhance management efficacy.