Achromobacter species are increasingly isolated from the respiratory tract of cystic fibrosis patients and often a chronic infection is established. How Achromobacter sp. adapts to the human host remains uncharacterised. By comparing longitudinally collected isolates of Achromobacter sp. isolated from five CF patients, we have investigated the within-host evolution of clonal lineages. The majority of identified mutations were isolate-specific suggesting co-evolution of several subpopulations from the original infecting isolate. The largest proportion of mutated genes were involved in the general metabolism of the bacterium, but genes involved in virulence and antimicrobial resistance were also affected. A number of virulence genes required for initiation of acute infection were selected against, e.g. genes of the type I and type III secretion systems and genes related to pilus and flagellum formation or function. Six antimicrobial resistance genes or their regulatory genes were mutated, including large deletions affecting the repressor genes of an RND-family efflux pump and a beta-lactamase. Convergent evolution was observed for five genes that were all implicated in bacterial virulence. Characterisation of genes involved in adaptation of Achromobacter to the human host is required for understanding the pathogen-host interaction and facilitate design of future therapeutic interventions.