Elucidating mechanisms underlying community assembly and biodiversity patterns is central to ecology and evolution. Genome size (GS, i.e. nuclear DNA content) determines species’ capacity to tolerate environmental stress or to exploit new environments and therefore potentially drive community assembly. However, its role in driving β-diversity (i.e., the site-to-site variability in species composition) remains unclear. We measured GS for 169 plant species and investigated their occurrences within plant communities across 52 sites spanning a 3200-km transect in the temperate grasslands of China. We found environmental factors showed larger effects on β-diversity of large-GS than that of small-GS species. Community weighted mean GS increased with mean annual precipitation, soil total nitrogen and phosphorus concentrations, but decreased with mean annual temperature, suggesting a negative selection against species with large GS in resources-limited or warmer climates. These findings highlight the roles for GS in driving community assembly and predicting species responses to climate change.