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In the Amazon basin, the distribution of many vertebrate species is delimited by large rivers, which are frequently considered as biogeographical barriers strongly related to the origin and maintenance of the elevated biodiversity found in the region. In this study, we conducted a phylogeographical investigation of the effect of the Tapajós River on multiple classes of genotypic and phenotypic characters in a species of frog, Allobates tapajos. We sampled populations throughout the known distributional range of the species on both margins of the middle and lower sections of the river. We obtained fragments of mitochondrial (16S) and nuclear (RAG1) genes, as well as external morphometric measurements and advertisement call acoustic parameters of 48 individuals from six localities (populations). While the nuclear marker was monomorphic across the geographic distribution of A. tapajos, the mitochondrial fragment revealed low genetic distances accompanied by high spatial structuring, with restricted and absent haplotype sharing between populations and opposite river margins, respectively. Cladogenetic events were concentrated in the Pleistocene epoch, the time period corresponding to the establishment of the Tapajós River drainage. Acoustic parameters diverged between river margins, a pattern not observed in relation to the morphological markers analysed. There was no correlation in the variability pattern of the different classes of characters between them or in relation to linear geographic distance among populations. In addition, discriminant function analyses correctly assigned most of the individuals to their populations based on phenotypic characters. Our results show that the distribution of the variability within A. tapajos is affected not only by the transposition of a historical riverine barrier but also mostly by an elevated genotypic and phenotypic structure at the population level. [ABSTRACT FROM AUTHOR]