Disentangling the impact of landscape features such as rivers and historical events on dispersal is a challenging but necessary task to gain a comprehensive picture of the evolution of diverse biota such as that found in Amazonia. Adenomera andreae, a small, territorial, terrestrial frog species of the Amazonian forest represents a good model for such studies. We combined Cytochrome b sequences with 12 microsatellites to investigate the genetic structure at two contrasted spatial scales in French Guiana: along a ~6 km transect to evaluate dispersal ability and between paired bank populations along a ~65 km stretch of the Approuague river to test the effect of rivers as barriers to dispersal. We observed significant spatial genetic structure between individuals at a remarkably small geographical scale and conclude that the species has a restricted dispersal ability likely tied to its life history traits. Mitochondrial and microsatellite data also indicate a high level of differentiation among populations on opposite banks of the river and, in some cases, among populations on the same riverbank. These results suggest that the observed population structure in A. andreae is due to restricted dispersal abilities combined with the action of rivers and Quaternary population isolation. Given that Amazonia hosts a great portion of anurans, as well as other small vertebrates, that display life history traits comparable to Adenomera andreae, we argue that our analyses provide new insights into the complex interactions among evolutionary processes shaping Amazonian biodiversity.