Plants intimately associate with diverse bacteria. Plant-associatedbacteria have ostensibly evolved genes that enable them to adapt to plantenvironments. However, the identities of such genes are mostly unknown, and theirfunctions are poorly characterized. We sequenced 484 genomes of bacterial isolatesfrom roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterialgenomes to identify thousands of plant-associated gene clusters. Genomes ofplant-associated bacteria encode more carbohydrate metabolism functions and fewermobile elements than related non-plant-associated genomes do. We experimentallyvalidated candidates from two sets of plant-associated genes: one involved in plantcolonization, and the other serving in microbe–microbe competition betweenplant-associated bacteria. We also identified 64 plant-associated protein domainsthat potentially mimic plant domains; some are shared with plant-associated fungiand oomycetes. This work expands the genome-based understanding of plant–microbeinteractions and provides potential leads for efficient and sustainable agriculturethrough microbiome engineering.