Abstract Major historical landscape changes have left significant signatures on species diversification. However, how these changes have affected the build-up and maintenance of Amazonia’s megadiversity continues to be debated. Here, we addressed this issue by focusing on the evolutionary history of a pan-Amazonian toad genus that has diversified throughout the Neogene (Amazophrynella). Based on a comprehensive spatial and taxonomic sampling (286 samples, all nominal species), we delimited operational taxonomic units (OTUs) from mitochondrial DNA sequences. We delimited 35 OTUs, among which 13 correspond to nominal species, suggesting a vast underestimation of species richness. Next, we inferred time-calibrated phylogenetic relationships among OTUs based on complete mitogenomic data, which confirmed an ancient divergence between two major clades distributed in eastern and western Amazonia, respectively. Ancestral area reconstruction analyses suggest that the Andean foothills and the Brazilian Shield region represent the ancient core areas for their diversification. These two clades, probably isolated from one other by lacustrine ecosystems in western Amazonia during the Miocene, display a pattern of northward and eastward dispersals throughout the Miocene‒Pliocene. Given the ecological association of Amazophrynella with non-flooded forests, our results reinforce the perception that ancient Amazonian landscape changes had a major impact on the diversification of terrestrial vertebrates.