Aim: We investigate the spatiotemporal context of the diversification of Allobates, a widespread genus of Amazonian frogs with high species diversity particularly in western Amazonia. We tested if that diversity originated in situ or through repeated dispersals from other Amazonian areas and if this diversification took place during or after the Pebas system, a vast lacustrine system occupying most western Amazonia between 23 and 10 million years ago (Mya). Location: Amazonia. Taxon: Allobates (Anura: Aromobatidae). Methods: We gathered a spatially and taxonomically extensive sampling of mitochondrial DNA sequences from 932 Allobates specimens to delimit Operational Taxonomic Units (OTUs). Complete mitogenomes of these OTUs were assembled to reconstruct a time-calibrated phylogeny used to infer the historical and spatial origin of the Amazonian Allobates lineages. Results: Phylogenetic analyses and ancestral area reconstruction suggested that most of the western Amazonian lineages resulted from in situ diversification and that these events occurred between the inferred origin of the genus (25 Mya) and 10 Mya, with a possible peak between 14 and 10 Mya. Dispersal among areas mostly occurred from western Amazonia towards the Brazilian and the Guiana Shields. Closely related Allobates OTUs display an allopatric pattern of distribution, matching interfluves delimited by modern Amazonian rivers. Main Conclusions: In western Amazonia, diversification of Allobates appears to have been simultaneous with the last stages of the Pebas system (14–10 Mya). Subsequently (within the last 10 Mya), modern Amazonian river courses shaped the distribution pattern of Allobates species and possibly promoted allopatric speciation.