River ecosystems have a key role in the processes, which control the storage of atmospheric CO2. However, because of strong spatial and temporal variabilities in the sources of organic matter (OM), their relative contribution to the carbon dynamics of large river ecosystems is not fully understood. The present work aimed to characterize the sources of OM in the central Amazon basin. The fatty acids (FA) and stable carbon and nitrogen isotopic (δ13C, δ15N) compositions of OM were investigated in five floodplains (locally called Várzea), the Amazon River and four of its main tributaries (Solimões, Negro, Madeira and Tapajós). The OM analyzed includes macrophytes (Eichornia sp., Paspalum repens, Pistia stratiotes and Salvinia auriculata), plankton samples, suspended particulate organic matter (SPOM), sediments and soils from the flooded forest. A seasonal characterization of this OM, according to the annual oscillation in the hydrological cycle, indicates a strong autotrophic production from cyanobacteria in Várzea during the drought season. At this season, a transfer of OM, resulting from cyanobacteria and decomposition of macrophytes, has been evidenced from the Várzea to the rivers. However, these plants, which use different pathways of photosynthesis, do not contribute equally to OM and to the food web. Thus, a degradation experiment showed the decomposition dynamics of these macrophytes and the role of micro-organisms. The characterization of OM sources, their spatial and seasonal variabilities, the processes involved in its degradation as well as its utilization by consumers, indicate that the OM produced in Várzea constitute the major source of respired carbon in rivers from the Amazon basin and results from phytoplanktonic production.