Among the hypotheses formulated to explain the origin of Amazonian biodiversity, two (the riverine-barrier and the river-refuge hypotheses) focus on the role that rivers play as biotic barriers promoting speciation. However, empirical results have both supported and refuted these hypotheses. This is likely due, at least in part, to river-specific hydrologic characteristics and the biology of the focal species. The rivers of the Guiana Shield represent a model system because they have had more stable courses over time than those of the western Amazon Basin, where most tests of riverine barrier effects have taken place. We tested whether life-history traits (body size, habitat and larval development), expected to be important in determining dispersal ability, of 28 frog species are associated with genetic structure and genetic distances of individuals sampled from both banks of the Oyapock River. Thirteen of these species displayed genetic structure consistent with the river acting as a barrier to dispersal. Surprisingly, body size was not correlated with trans-riverine population structure. However, leaf-litter dwellers and species lacking free-living tadpoles were found to exhibit higher river-associated structure than open habitat/arboreal species and those with exotrophic tadpoles. These results demonstrate that rivers play an important role in structuring the genetic diversity of many frog species though the permeability of such riverine barriers is highly dependent on species-specific traits.