Lotic ecosystems are sources of greenhouse gases (GHGs) to the atmosphere, but their emissions are uncertain due to longitudinal GHG heterogeneities associated with point source pollution from anthropogenic activities. In this study, we quantified summer concentrations and fluxes of carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), and dinitrogen (N2), as well as several water quality parameters along the Rhine River and the Mittelland Canal, two critical inland waterways in Germany. Our main objectives were to compare GHG concentrations and fluxes along the two ecosystems and to determine the main driving factors responsible for their longitudinal GHG heterogeneities. The results indicated that the two ecosystems were sources of GHG fluxes to the atmosphere, with the Mittelland Canal being a hotspot for CH4 and N2O fluxes. We also found significant longitudinal GHG flux discontinuities along the mainstems of both ecosystems, which were mainly driven by divergent drivers. Along the Mittelland Canal, peak CO2 and CH4 fluxes coincided with point pollution sources such as a joining river tributary or the presence of harbors, while harbors and in-situ biogeochemical processes such as methanogenesis and respiration mainly explained CH4 and CO2 hotspots along the Rhine River. In contrast to CO2 and CH4 fluxes, N2O longitudinal trends along the two lotic ecosystems were better predicted by in-situ parameters such as chlorophyll-a concentrations and N2 fluxes. Based on a positive relationship with N2 fluxes, we hypothesized that in-situ denitrification was driving N2O hotspots in the Canal, while a negative relationship with N2 in the Rhine River suggested that coupled biological N2 fixation and nitrification accounted for N2O hotspots. These findings stress the need to include N2 flux estimates in GHG studies, as it can potentially improve our understanding of whether nitrogen is fixed through N2 fixation or lost through denitrification.