Andes-to-Amazon river connectivity controls numerous natural and human systems in the greater Amazon. However, it is being rapidly altered by a wave of new hydropower development, the impacts of which have been previously underestimated. We document 142 dams existing or under construction and 160 proposed dams for rivers draining the Andean headwaters of the Amazon. Existing dams have fragmented the tributary networks of six of eight major Andean Amazon river basins. Proposed dams could result in significant losses in river connectivity in river mainstems of five of eightmajor systems-the Napo, Marañón, Ucayali, Beni, andMamoré.With a newly reported 671 freshwater fish species inhabiting the Andean headwaters of the Amazon (>500 m), dams threaten previously unrecognized biodiversity, particularly among endemic andmigratory species. Because Andean rivers contribute most of the sediment in the mainstem Amazon, losses in river connectivity translate to drastic alteration of river channel and floodplain geomorphology and associated ecosystem services.