There is currently limited understanding of the contribution of biological N2 fixation (diazotrophy) to the N budget of large river systems. This natural source of N in boreal river systems may partially explain the sustained productivity of river floodplains in Northern Europe where winter fodder was harvested for centuries without fertilizer amendments. In much of the world, anthropogenic pollution and river regulation have nearly eliminated opportunities to study natural processes that shaped early nutrient dynamics of large river systems; however, pristine conditions in northern Fennoscandia allow for the retrospective evaluation of key biochemical processes of historical significance. We investigated biological N2 fixation (diazotrophy) as a potential source of nitrogen fertility at 71 independent floodplain sites along 10 rivers and conducted seasonal and intensive analyses at a subset of these sites. Biological N2 fixation occurred in all floodplains, averaged 24.5 kg N ha-1 yr-1 and was down regulated from over 60 kg N ha-1 yr-1 to 0 kg N ha-1 yr -1 by river N pollution. A diversity of N2-fixing cyanobacteria was found to colonize surface detritus in the floodplains. The data provide evidence for N2 fixation to be a fundamental source of new N that may have sustained fertility at alluvial sites along subarctic rivers. Such data may have implications for the interpretation of ancient agricultural development and the design of contemporary low-input agroecosystems.