Stream networks have recently been discovered to be major but poorly constrained natural greenhouse gas (GHG) sources. A fundamental problem is that several measurement approaches have been used without cross-comparisons. Flux chambers represent a potentially powerful methodological approach if robust and reliable ways to use chambers on running water can be defined. Here we compare the use of anchored and freely drifting chambers on various streams with different flow velocities. The study clearly shows that (1) anchored chambers enhance turbulence under the chambers and thus elevate fluxes, (2) drifting chambers have a very small impact on the water turbulence under the chamber and thus generate more reliable fluxes, (3) the bias of the anchored chambers greatly depends on chamber design and sampling conditions, and (4) there is a promising method to reduce the bias from anchored chambers by using a flexible plastic foil collar to seal the chambers to the water surface, rather than having rigid chamber walls penetrating into the water. Altogether, these results provide novel guidance on how to apply flux chambers in running water, which will have important consequences for measurements to constrain the global GHG balances.