The nature of structuring phenomena potentially responsible for the diversification of Amazonian fish fauna during the Neogene has been explored through a comparative phylogeography strategy relying on mitochondrial DNA sequences. Six species with contrasted life history traits, and belonging to three representative Amazonian families were sampled with an emphasis in the Upper Madera: Colossoma macropomum, Piaractus brachypomus, Pygocentrus nattereri, Pseudoplatystoma punctifer, P. tigrinum and Cichla pleiozona. At the macroevolutionary scale, the role played by major geomorphological transformations was corroborated. While at the microevolutionary scale most of the inferred populations were found to be far from the migration-drift equilibrium and beard genetic signals of significant demographic transformations. We propose that some specific events may have affected the bulk of the fish fauna of the Upper Madera region (Bolivian Amazon) during the latest part of the Pleistocene. The particular biological nature of the Upper Madera urges the need for conservation studies and science based management policies.