Conservation practitioners increasingly recognize the conservation value of tributaries for supporting mainstem, large‐river specialist fishes. A tributary’s discharge at its mouth is a coarse indicator of the richness of large‐river specialists found within the tributary, but the relative influences of regional dispersal and local habitat underpinning this species–discharge relationship are often unknown. We sampled large‐river specialist fishes at sites within two nonwadeable tributaries of the Missouri and Mississippi rivers with contrasting prairie (Grand River) vs. upland (Meramec River) habitats to address four research questions: (1) Do alpha diversity (mean site‐level species richness) and beta diversity (among‐site species compositional differences) vary between tributaries? (2) Does mean annual discharge correlate with local habitat and downstream distance to mainstem rivers (i.e., mainstem connectivity)? (3) Are slopes of species–discharge relationships consistent between tributaries? And (4) Do local habitat and downstream distance explain residual richness at sites beyond variation already explained by species–discharge relationships? We detected 30 of 42 potential large‐river specialist fishes, demonstrating most mainstem species use tributaries. Mean site richness was higher in the Grand River (12.5 species vs. 9.8 species in Meramec River), but partitioning of lower reaches (sites
