Hydropower expansion is increasingly responsible for connectivity and biodiversity loss in freshwater ecosystems. The Amazon basin, which supports the highest level of freshwater biodiversity globally, faces such unplanned expansion. Here, we demonstrate and quantify the impacts of two major hydroelectric dams on the Madeira River on the trans‐Amazonian movements of one apex predatory catfish ( Brachyplatystoma rousseauxii ) performing the longest migration known in freshwaters. Using otolith microchemistry, we show that above the dams, the former basin‐wide homing migration between the breeding grounds in the upper Madeira and the nursery in the Amazon estuary has now been replaced by residency. We found evidence suggesting downstream migration of juvenile fish past dams into the Amazon estuary and that some of them home back to the Madeira. However, we did not find evidence that the fish homing back from the estuary can access their breeding grounds in the upper Madeira; they remain blocked below the dams. Our results provide undisputable evidence that the conservation of the species primarily requires the reestablishment of connectivity with the construction of efficient fishways.