Cross-infection by Pseudomonas aeruginosa between unrelated patients with cystic fibrosis (CF) is believed to be uncommon. After detecting a genotypically identical strain of P. aeruginosa in five unrelated children with CF dying from severe lung disease, we determined its prevalence within a large CF clinic using pulsed-field gel electrophoresis and random amplified polymorphic DNA assays. The clinical status of P. aeruginosa–infected patients was also determined. Between September and December 1999, 152 patients, aged 3.9–20.7 years, provided sputum for culture. P. aeruginosa was detected in 118 children of mean (SD) age 13.5 (3.8) years. The genotyping techniques were concordant, showing that 65 (55%) infected patients carried an indistinguishable or closely related strain. No distinctive antibiogram or environmental reservoir was found. Patients with the clonal strain were more likely than those with unrelated isolates to have been hospitalized in the preceding 12 months for respiratory exacerbations. This study demonstrates extensive spread of a single, clonal strain of P. aeruginosa in a large pediatric CF clinic. Whether this strain is also more virulent than sporadic isolates remains to be determined. As transmissible strains could emerge elsewhere, other CF clinics may also need to consider molecular methods of surveillance for cross-infection.