Rationale: During long-term lung infection in cystic fibrosis patients, Pseudomonas aeruginosa strains develop mutations leading to clonal expansion. This microevolution is thought to be correlated with a reduced virulence. Objectives: We tested this hypothesis in models of lung infection, using mice with different genetic backgrounds. Methods: From infected airways of six cystic fibrosis patients, 25 P. aeruginosa clones were isolated during a period of up to 16.3 years and genotypically and phenotypically characterized. Virulence of the eight early, six intermediate and 11 late cystic fibrosis isolates and five environmental strains was assessed by monitoring acute mortality versus survival and P. aeruginosa chronic persistence versus lung clearance in mice of different genetic backgrounds, including cystic fibrosis mice. Measurements and main results: Different patients harboured clonally unrelated strains, but early, intermediate and late P. aeruginosa isolates from single patients were clonally related, allowing comparative in vivo analysis. While late isolates were attenuated in causing acute mortality in the mouse models, compared to early and intermediate clonal isolates and environmental strains, they did not differ from early and intermediate clonal isolates in their capacity to establish chronic infection and cause extensive inflammation in the murine respiratory tract. Conclusion: Our findings indicate that clonal expansion of P. aeruginosa strains during microevolution within cystic fibrosis lungs leads to populations with altered but not reduced virulence. These P. aeruginosa clones with adapted virulence play a critical role in the pathogenesis of chronic infections and may serve to define virulence determinants as targets for novel therapies.