Methane (CH4) fluxes from world rivers are still poorly constrained, with measurements restricted mainly to temperate climates. Additional river flux measurements, including spatio-temporal studies, are important to refine extrapolations. Here we assess the spatio-temporal variability of CH4 fluxes from the Amazon and its main tributaries, the Negro, Solimões, Madeira, Tapajós, Xingu, and Pará Rivers, based on direct measurements using floating chambers. Sixteen of 34 sites were measured during low and high water seasons. Significant differences were observed within sites in the same river and among different rivers, types of rivers, and seasons. Ebullition contributed to more than 50% of total emissions for some rivers. Considering only river channels, our data indicate that large rivers in the Amazon Basin release between 0.40 and 0.58 Tg CH4 yr−1. Thus, our estimates of CH4 flux from all tropical rivers and rivers globally were, respectively, 19–51% to 31–84% higher than previous estimates, with large rivers of the Amazon accounting for 22–28% of global river CH4 emissions.