Burkholderia multivorans strains from 47 cystic fibrosis (CF) patients in 28 hospitals were compared by pulsed-field gel electrophoresis (PFGE) and flagellin (fliC) PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. A considerable degree of genetic variation was evident, with each patient harboring a strain with a unique PFGE profile. Four sizes of fliC amplicons were produced, and these amplicons gave 13 RFLP types with restriction enzyme MspI. B. multivorans did not appear to spread between patients, suggesting that most CF patients acquire the organism from the natural environment.