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 and therefore potentially drives community assembly. However, its role in driving β-diversity (i.e., spatial variability in species composition) remains unclear. We measured GS for 161 plant species and investigated their occurrences within plant communities across 52 sites spanning a 3200-km transect in the temperate grasslands of China. Using species distribution modelling, we found that environmental factors showed larger effects on β-diversity of large-GS than that of small-GS species and that communities with abundant resources had a greater representation of large-GS species. The latter finding was confirmed following analysis of data from a 10-yr resource (water, nitrogen, and phosphorus) manipulation experiment in which resource addition resulted in increased community weighted GS based on plant biomass estimates, suggesting that large-GS species are more sensitive to environmental resource limitation and explaining the greater environmental selection on β-diversity of large-GS species. These findings highlight the roles of GS in driving community assembly and predicting species responses to global change.