Pseudomonas aeruginosa is an opportunistic bacterial pathogen commonly associated with chronic lung infection in cystic fibrosis (CF). To better understand the molecular mechanisms underpinning adaptation of the bacteria to the CF lung microenvironment we conducted genomic, proteomic and phenotypic characterisation of four novel CF isolates of P. aeruginosa cultured in various growth media (Synthetic Cystic Fibrosis Media, Luria-Bertani (LB) broth, M9-Glucose) and under oxygen stress (O2<1%). Genomic and phenotypic analyses revealed significant diversity in colonization, virulence and metabolic traits different to a reference laboratory strain PAO1. Whole cell and membrane proteome profiling using combinations of iTRAQ, TMT and SWATH mass spectrometry enabled quantification of 3,849 proteins from whole cell extracts and 990 membrane proteins from membrane enriched fractions (FDR <1%) mapping around 71% of predicted ORFs and representing the most comprehensive proteome of clinically relevant P. aeruginosa reported to date. CF clinical isolates shared a core proteomic signature which is different to PAO1. In comparison to PAO1, we observed down-regulation of motility, adhesion and chemotaxis proteins (FliK, PilJ, PctA), questioning the utility of targeting these molecules for vaccine development. We also saw up-regulation of proteins used in respiration (Cbb3-1,Cbb3-2,NarG-I, NirC, NorB) and drug resistance (MexY, MexB, MexC). Functional assays including antibiotic MIC assay, motility and CF sputum adhesion assays confirmed the proteomic findings. These proteome maps and phenotypic profiles illuminate the diversity in the adaptation and micro-evolution mechanisms of P. aeruginosa that cannot be detected at the genome level alone.