The reciprocal interaction between rhizosphere bacteria and their plant hosts results in a complex battery of genetic and physiological responses. In this study, we used insertion sequencing (INSeq) to reveal the genetic determinants responsible for the fitness of Pseudomonas aeruginosa PGPR2 during root colonization. We generated a random transposon mutant library of Pseudomonas aeruginosa PGPR2 comprising 39,500 unique insertions and identified genes required for growth in culture and on corn roots. A total of 108 genes were identified as contributing to the fitness of strain PGPR2 on roots. The importance in root colonization of four genes identified in the INSeq screen was verified by constructing deletion mutants in the genes and testing them for the ability to colonize corn roots singly or in competition with the wild type. All four mutants were affected in corn root colonization, displaying 5- to 100-fold reductions in populations in single inoculations, and all were outcompeted by the wild type by almost 100-fold after seven days on corn roots in mixed inoculations of the wild type and mutant. The genes identified in the screen had homology to genes involved in amino acid catabolism, stress adaptation, detoxification, signal transduction, and transport. INSeq technology proved a successful tool to identify fitness factors in P. aeruginosa PGPR2 for root colonization.