Most knowledge about evolutionary adaptation has been gained from experimental evolution studies, in which organisms have been allowed to evolve under simple, well-defined conditions in the laboratory. While these studies have provided novel insight into the fundamental processes of evolutionary adaptation, it is less clear to which extent the observations can be generalized to natural systems, in which organisms evolve in complex heterogeneous environments. The focus of this thesis has been to explore different aspects of evolutionary adaptation of bacterial populations evolving in natural environments. The model system used for these investigations has been long-term chronic airway infections in Cystic fibrosis (CF) patients caused by the opportunistic pathogen Pseudomonas aeruginosa. Using a systems biology approach, we have monitored the adaptive development of the clinically important P. aeruginosa DK2 clone lineage during 200,000 generations of evolution in the CF airways from its entrance in the clinic in the 1970’ies until the end of 2010. Genetic analysis showed that the DK2 lineage between 1973 and 2007 accumulated mutations in a near-linear manner with an overall genomic signature of negative selection. Phenotypic profiling (gene expression and catabolic performance) showed that major phenotypic changes occurred in the DK2 lineage during the early years until 1979, after which only marginal phenotypic changes could be observed in and between isolates. Through the use of genetic reconstructions it was shown that many of the phenotypic changes were caused by mutations in genes encoding regulators of large regulatory networks in P. aeruginosa. Moreover, it was shown that the combination of mutations gave rise to unexpected CF-adaptive phenotypes such as increased tolerance to antibiotics as well as a conditional mucoid phenotype which was induced only upon exposure to CF-associated stress conditions such as high osmolarity and anaerobic conditions. Appearance of a constitutive mucoid phenotype was discovered during the late stages of the DK2 colonization in a specific CF patient. The mucoid DK2 isolates emerged in connection with a prolonged period of non-compliance to antibiotic treatment and correlated with a permanent increase in the inflammatory response. Genetic analysis shows that several mucoid variants evolved in parallel by distinct mutational pathways; one of these pathways included fixation of mutations in the rpoD gene encoding the principle sigma factor σ70. The findings presented in this thesis provide insight into the genetic mechanisms and evolutionary processes that shape the adaptation of bacteria colonizing complex natural environments. Increased knowledge of the evolutionary trajectories of pathogens may lead to significant improvements in the future treatment of patients suffering from severe chronic infections.