Large rivers transport considerable amounts of terrestrial dissolved organic matter (DOM) to the ocean. Yet, downstream gradients and temporal variability in DOM fluxes and characteristics are poorly studied at the scale of large river basins, especially in tropical areas. Here, we report longitudinal patterns in DOM content and composition based on absorbance and fluorescence measurements along the Zambezi River and its main tributary, the Kafue River, during two hydrological seasons. During high flow periods, a greater proportion of aromatic and humic DOM was mobilized along rivers due to the hydrological connectivity with wetlands and high flow velocities, while low flow periods were characterized by lower DOM content of less aromaticity resulting from loss of connectivity with wetlands, more efficient degradation of terrestrial DOM and enhanced autochthonous productivity. Changes in water residence time due to contrasting water discharge were found to modulate the fate of DOM along the river continuum. Thus, terrestrial DOM dynamics shifted from transport-dominated during the wet seasons towards degradation during the dry season, with substantial consequences on longitudinal DOM content and composition. The longitudinal evolution of DOM was also strongly impacted by a hydrological buffering effect in large reservoirs in which the seasonal variability of DOM fluxes and composition was strongly reduced.