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Pseudomonas aeruginosa is an opportunistic pathogen found ubiquitously in the environment and commonly associated with airway infection in patients with cystic fibrosis. P. aeruginosa strain PAO1 is one of the most commonly used laboratory-adapted research strains and is a standard laboratory-adapted strain in multiple laboratories and strain banks worldwide. Due to potential isolate-to-isolate variability, we investigated the genomic and phenotypic diversity among 10 PAO1 strains (henceforth called sublines) obtained from multiple research laboratories and commercial sources. Genomic analysis predicted a total of 5,682 genes, with 5,434 (95.63%) being identical across all 10 strains. Phenotypic analyses revealed comparable growth phenotypes in rich media and biofilm formation profiles. Limited differences were observed in antibiotic susceptibility profiles and immunostimulatory potential, measured using heat-killed whole-cell preparations in four immortalized cell lines followed by quantification of interleukin-6 (IL-6) and IL-1β secretion. However, variability was observed in the profiles of secreted molecular products, most notably, in rhamnolipid, pyoverdine, pyocyanin, Pseudomonas quinolone signal (PQS), extracellular DNA, exopolysaccharide, and outer membrane vesicle production. Many of the observed phenotypic differences did not correlate with subline-specific genetic changes, suggesting alterations in transcriptional and translational regulation. Taken together, these results suggest that individually maintained sublines of PAO1, even when acquired from the same parent subline, are continuously undergoing microevolution during culture and storage that results in alterations in phenotype, potentially affecting the outcomes of in vitro phenotypic analyses and in vivo pathogenesis studies. IMPORTANCE Laboratory-adapted strains of bacteria are used throughout the world for microbiology research. These prototype strains help keep research data consistent and comparable between laboratories. However, we have observed phenotypic variability when using different strains of Pseudomonas aeruginosa PAO1, one of the major laboratory-adopted research strains. Here, we describe the genomic and phenotypic differences among 10 PAO1 strains acquired from independent sources over 15 years to understand how individual maintenance affects strain characteristics. We observed limited genomic changes but variable phenotypic changes, which may have consequences for cross-comparison of data generated using different PAO1 strains. Our research highlights the importance of limiting practices that may promote the microevolution of model strains and calls for researchers to specify the strain origin to ensure reproducibility., (Copyright © 2019 American Society for Microbiology.)