Background <p><i>Pseudomonas aeruginosa</i> is a major cause of acute nosocomial infections, as well as chronic respiratory infections associated with cystic fibrosis (CF). In chronic lung infections, <i>P. aeruginosa</i> populations typically exhibit extensive phenotypic variation, a trait linked to their need to undergo pathoadaptive mutations to counteract host-derived selective pressures.</p> Methods <p>In this study, two clonally related <i>P. aeruginosa</i> isolates, SCPA07 and SCPA08, were identified to coexist in a single bronchoalveolar lavage fluid (BALF) sample from a patient with bacterial pneumonia. Whole-genome sequencing (WGS) was conducted to characterize their genetic background, as well as antimicrobial resistance and virulence gene profiles. A comprehensive analysis of phylogenetic relationships and comparative genomic features of the two isolates was conducted using a panel of bioinformatics tools. Their antimicrobial resistance mechanisms were elucidated via gene sequence analysis and quantitative reverse-transcription PCR (qRT-PCR). A series of phenotypic experiments, including growth, biofilm formation, environmental stress, and virulence assays, and others were performed to characterize their phenotypic traits.</p> Results <p>Antimicrobial susceptibility assay showed that both strains were carbapenem-non-susceptible (defined as intermediate or resistant according to the Clinical and Laboratory Standards Institute (CLSI) guidelines). Genomic analysis revealed that they are ‘hypermutator’ strains and harbor both the <i>exoS</i> and <i>exoU</i> virulence genes, indicating an increased propensity for persistent host infection and high virulence potential. Both strains harbored mutations in <i>oprD</i>, and exhibited elevated expression of AmpC β-lactamase and the efflux pump MexB, which most likely contributed to their non-susceptibility to carbapenems. Despite harboring nucleotide variations at only ten genetic loci, these two strains exhibited distinct phenotypic traits: SCPA07 showed rapid growth, strong biofilm formation, high virulence, and growth advantages under iron limitation and serum stress; in contrast, its variant SCPA08 had slow growth, poor motility, reduced pyocyanin production, low virulence, and increased tolerance to the host antimicrobials human cathelicidin LL-37 and hydrogen peroxide (H₂O₂). These phenotypic variations are proposed to be primarily driven by genetic mutations affecting the O-antigen biosynthesis, iron utilization, Porin D, and other determinants.</p> Conclusions <p>This study elucidates the divergent adaptive evolutionary strategies of a single <i>exoS</i>⁺/<i>exoU</i>⁺ <i>P. aeruginosa</i> clone within the host during bacterial pneumonia, as well as their critical role in shaping the bacterium’s virulence and adaptability, which sheds light on the within-host evolution dynamics of <i>P. aeruginosa</i> populations during their pathogenesis and persistence in the lung.</p>