The biogeographical distribution of methanotrophic communities and the mechanisms driving methanotrophic community assembly in grassland soils are largely unknown. In this study, the assembly mechanisms of methanotrophic communities and the niche preferences of specific methanotrophic taxa were investigated in three Chinese grassland regions, including the Inner Mongolian Plateau (IMP), the Xinjiang Autonomous Region (XAR) and the Tibetan Plateau (TP) across 1500, 1000 and 1000 km, respectively. We analyzed the relative importance of deterministic and stochastic processes on methanotrophic community assembly and assessed how assembly is affected by environmental and spatial factors. The results revealed the presence of various methanotrophic taxa in the different grassland regions. Deterministic processes were dominant in the methanotrophic community assembly, and heterogeneous selection had a greater contribution in shifting the methanotrophic community than did homogeneous selection, dispersal limitations, homogenizing dispersal and drift. Moreover, key environmental factors shaping the methanotrophic communities were scale-dependent and varied among the three regions. The primary variables identified as shaping methanotrophic communities were the aridity index, total nitrogen, and mean annual temperature in the IMP, XAR and TP grasslands at regional scales, whereas pH was the primary variable when considering all of the regions together. In general, the soil methanotrophic community assembly was primarily influenced by deterministic processes and responded in a predictable manner to major environmental factors at a regional scale.