Summary Lake methane (CH 4 ) emissions are largely controlled by aerobic methane‐oxidizing bacteria (MOB) which mostly belong to the classes Alpha‐ and Gammaproteobacteria (Alpha‐ and Gamma‐MOB). Despite the known metabolic and ecological differences between the two MOB groups, their main environmental drivers and their relative contribution to CH 4 oxidation rates across lakes remain unknown. Here, we quantified the two MOB groups through CARD‐FISH along the water column of six temperate lakes and during incubations in which we measured ambient CH 4 oxidation rates. We found a clear niche separation of Alpha‐ and Gamma‐MOB across lake water columns, which is mostly driven by oxygen concentration. Gamma‐MOB appears to dominate methanotrophy throughout the water column, but Alpha‐MOB may also be an important player particularly in well‐oxygenated bottom waters. The inclusion of Gamma‐MOB cell abundance improved environmental models of CH 4 oxidation rate, explaining part of the variation that could not be explained by environmental factors alone. Altogether, our results show that MOB composition is linked to CH 4 oxidation rates in lakes and that information on the MOB community can help predict CH 4 oxidation rates and thus emissions from lakes.