This study investigates adaptation of ST406, a prevalent P. aeruginosa clone, present in 15% of chronically infected cystic fibrosis (CF) patients in the Netherlands, in a newly infected CF patient during three years using whole genome sequencing (WGS), transcriptomics, and phenotypic assays, including biofilm formation. WGS-based phylogeny demonstrates that ST406 is genetically distinct from other reported CF related strains or epidemic clones. Comparative genomic analysis of the early (S1) and late (S2) isolate yielded 42 single nucleotide polymorphisms (SNPs) and 10 indels and a single 7 kb genomic fragment only found in S2. Most SNPs and differentially expressed genes encoded proteins involved in metabolism, secretion and signal transduction or transcription. SNPs were identified in regulator genes mexT and exsA and coincided with differential gene expression of mexE and mexF, encoding the MexE/F efflux pump, genes encoding the type six secretion system (T6SS) and type three secretion system (T3SS), which have also been previously implicated in adaptation of other P. aeruginosa strains during chronic infection of CF lungs. The observation that genetically different strains from different patients have accumulated similar genetic adaptations supports the concept of adaptive parallel evolution of P. aeruginosa in chronically infected CF patients. Phenotypically, there was loss of biofilm maturation coinciding with a significant lower level of transcription of both bfmR and bfmS during chronic colonization. These data suggest that the high-prevalent Dutch CF clone ST406 displays adaptation to the CF lung niche, which involves a limited number of mutations affecting regulators controlling biofilm formation and secretion and genes involved in metabolism. These genes could provide good targets for anti-pseudomonal therapy.