Based on the contemporary coexistence theory developed from macroecology, the regional species pool, environmental processes, dispersal assembly and biotic interactions are considered to determine the assembly of a local community. However, we do not fully understand whether these kinds of mechanisms are all-important for the whole microbial community or for specific functional groups. Here we studied the underlying processes driving the β-diversity of soil-based diazotrophic communities across a large geographical distance in Chinese grasslands by considering the roles of species pools and ecological assembly processes. Results indicated that the species pool (γ-diversity) showed significant and positive correlations to the observed β-diversity of the diazotrophic community; that is, diazotrophic community turnover increased with the size of the species pool. Environmental filtering played a greater role than dispersal effects or biotic interactions on β-diversity. Biotic interactions drove the assemblage of diazotrophs by influencing the co-occurrence pattern of species. Overall, these results demonstrated the importance of the species pool and local community assembly processes in shaping diazotrophic β-diversity patterns in grassland soils.