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Eighty-nine Stenotrophomonas maltophilia isolates were recovered from 42 cystic fibrosis patients in a paediatric hospital of Rome, Italy, during a 17-months period.Twenty patients presented repeated episodes of S. maltophilia isolates, with 2 to7 isolates per patient. The antimicrobial susceptibilities of the strains showed that trimethoprim-sulfamethoxazole was the most active antibiotic, inhibiting more than 90% of the strains, followed by levofloxacin, with 62 out of 89 isolates being sensitive (69.7%). Genetic relatedness of S. maltophilia isolates was investigated by pulsed-field gel electrophoresis (PFGE). A total of 49 well-defined distinct XbaI PFGE profiles were identified, with 39 different PFGE types encountered.Twenty-one PFGE types were represented by multiple isolates, with the most frequent being PFGE type 4 (11 isolates), PFGE type 9 and 11 (7 isolates each), and PFGE type 3 (5 isolates). Persistence of S. maltophilia infection or colonization was identified in 17 patients with repeated episodes, while genetically distinct S. maltophilia isolates from the same patient was found in 6 cases. Genetically identical or highly related isolates from different patients were observed within 8 multiple strain PFGE types.This study revealed a high genetic heterogeneity among S. maltophilia isolates recovered from cystic fibrosis patients. All together these data seem to confirm the wide environmental distribution of this pathogen. Different items of acquisition were involved both outside and inside the nosocomial settings, with the crosstransmission observed among clinical isolates of S. maltophilia among different patients probably due by multiple independent acquisitions from the environment as a main mode of transmission.