We conducted a 12-month survey of anuran assemblages in two permanent lotic ecosystems (Preto and Boleiras rivers) and their adjacent habitats within the Cerrado biome in southeastern Brazil. The main goals were to understand patterns of spatial distribution, niche breadth, and niche overlap for syntopic anuran species, studying a large portion of the river longitudinal gradient (from first- to fourth-order sections). On the basis of microhabitat use by frogs, we tested the hypotheses that (1) spatial niche breadth would decrease or spatial niche overlap would increase in richer assemblages if competition for space was an important factor structuring the studied communities, (2) local species spatial niche breadth would relate positively to available microhabitat diversity in the riparian vegetation understory, and (3) species that were more plastic in microhabitat use would be able to occupy a greater number of river sections. Patterns of spatial niche overlap were not related to total number of coexisting species, possibly indicating that the spatial niche is not oversaturated, which makes competition for space unlikely. Microhabitat diversity along the riverside vegetation understory did not influence species spatial niche breadth. Species were plastic in microhabitat use but still did not use them randomly. Broader spatial niches were not indicative of a species ability to occupy a greater number of sites along the river's longitudinal gradient. These results corroborate those previously found for unconnected streams, indicating that anuran distribution is more influenced by specific preferences and plasticity than by migration opportunities.