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Several hypotheses have been used to explain diversification in the Neotropics. Integrating evolution with ecology extends the scope of testing the frameworks of these hypotheses. We test diversification hypotheses by integrating phylogeography and ecological niche models (ENMs) using the rodent Hylaeamys megacephalus (Azara's broad-headed oryzomys or large headed rice rat) of the Amazon and dry forests, as a model. We estimated divergence times, ancestral areas, diversification events, historical demography, haplotype sharing, and genetic distances based on the mitochondrial cytochrome b gene. We generated ENMs and tested for niche divergence between lineages; integrated genetic data to predict gene flow corridors; and projected paleodistributions for comparison with historical demography. We found high structuring in northern Amazonia on the left bank of the Amazon River, and less structure but secondary contact in southern Amazonia and dry forests. The Northern Amazonian lineage diverged from the other lineages through dispersal followed by vicariance due to the Amazon River about 1.35Mya, while the Southern Amazonian and Cerrado lineages diverged through dispersal about 0.78 Mya. Paleodistribution models revealed expansions of dry forest lineages consistent with the Refugia Hypothesis, but not retraction for the humid forest lineage, which were not congruent with historical demography data. Niche divergence was not supported for the Northern Amazonian lineage, whereas habitat corridors linking current lineages suggest environmental continuity to their distributions that is concordant with a riverine barrier. In contrast, niche divergence was supported between the Southern Amazonian and Cerrado lineages, indicating that isolation followed by ecological divergence likely acted on this diversification. The recent Amazon River barrier and ecological differentiation observed here will surely provide insight for future studies and hypotheses of biodiversity diversification in the Neotropics. Studies that integrate evolution and ecology promise to disentangle alternative hypotheses and shed light on the biogeography of this megadiverse region.