Pseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS6100-induced coupled insertion-inversion mechanism. A selective advantage was created by insertion of IS6100 into wbpM, pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.