The patient-to-patient transmission of highly prevalent Pseudomonas aeruginosa clones which are associated with enhanced disease progression has led to strict segregation policies for cystic fibrosis (CF) patients in many countries. However, little is known about the population structure of P. aeruginosa among CF patients. The aim of the present cross-sectional study was to determine the prevalence and genetic relatedness of P. aeruginosa isolates from CF patients who visited two major CF centers in The Netherlands in 2007 and 2008. These patients represented 45% of the Dutch CF population. P. aeruginosa carriage in the respiratory tract was determined by standard microbiological culture techniques, and all phenotypically different isolates in the first specimens recovered in 2007 and 2008 were genotyped by multilocus sequence typing. A total of 313 (57%) of 551 patients whose samples were cultured carried P. aeruginosa. Two sequence types (STs), ST406 and ST497, were found in 15% and 5% of the patients, respectively, and 60% of the patients harbored a strain that was also found in at least two other patients. The risk ratios for carrying ST406 and ST497 were 17.8 (95% confidence interval [CI], 7.2 to 43.6) for those aged between 15 and 24 years and 6 (95% CI, 1.4 to 26.1) for those aged >25 years. ST406 and ST497 were not genetically linked to previously described epidemic clones, which were also not found in this CF population. The population structure of P. aeruginosa in Dutch CF patients is characterized by the presence of two prevalent STs that are associated with certain age groups and that are not genetically linked to previously described epidemic clones.