['To determine whether highly prevalent P. aeruginosa sequence types (ST) in Dutch cystic fibrosis (CF) patients are specifically linked to CF patients we investigated the population structure of P. aeruginosa from different clinical backgrounds. We first selected the optimal genotyping method by comparing pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and multilocus variable number tandem-repeat analysis (MLVA).', 'Selected P. aeruginosa isolates (n = 60) were genotyped with PFGE, MLST and MLVA to determine the diversity index (DI) and congruence (adjusted Rand and Wallace coefficients). Subsequently, isolates from patients admitted to two different ICUs (n = 205), from CF patients (n = 100) and from non-ICU, non-CF patients (n = 58, of which 19 were community acquired) were genotyped with MLVA to determine distribution of genotypes and genetic diversity.', 'Congruence between the typing methods was >79% and DIs were similar and all >0.963. Based on costs, ease, speed and possibilities to compare results between labs an adapted MLVA scheme called MLVA9-Utrecht was selected as the preferred typing method. In 363 clinical isolates 252 different MLVA types (MTs) were identified, indicating a highly diverse population (DI \u200a= 0.995; CI \u200a= 0.993-0.997). DI levels were similarly high in the diverse clinical sources (all >0.981) and only eight genotypes were shared. MTs were highly specific (>80%) for the different patient populations, even for similar patient groups (ICU patients) in two distinct geographic regions, with only three of 142 ICU genotypes detected in both ICUs. The two major CF clones were unique to CF patients.'] Does the population genetics of Pseudomonas aeruginosa isolates from different patient populations exhibit high-level host specificity?