The authors have developed a comprehensive transposon mutant library of *Pseudomonas aeruginosa* PAO1, a pathogen with a 6.3-Mbp genome. The library consists of 30,100 sequence-defined mutants, with an average of five insertions per gene. About 12% of the predicted genes lack insertions, many of which are likely essential for growth on rich media. Statistical and bioinformatic analyses estimate that the actual number of essential genes is between 300 and 400. The library was screened for mutants defective in twitching motility and prototrophic growth, identifying nearly all genes expected from earlier studies. The transposons used to generate the mutant collection have features that facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering. The library provides a valuable resource for future research and functional insights into the genome of *P. aeruginosa*.The authors have developed a comprehensive transposon mutant library of *Pseudomonas aeruginosa* PAO1, a pathogen with a 6.3-Mbp genome. The library consists of 30,100 sequence-defined mutants, with an average of five insertions per gene. About 12% of the predicted genes lack insertions, many of which are likely essential for growth on rich media. Statistical and bioinformatic analyses estimate that the actual number of essential genes is between 300 and 400. The library was screened for mutants defective in twitching motility and prototrophic growth, identifying nearly all genes expected from earlier studies. The transposons used to generate the mutant collection have features that facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering. The library provides a valuable resource for future research and functional insights into the genome of *P. aeruginosa*.