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Persistent methicillin-resistant Staphylococcus aureus (MRSA) infection in cystic fibrosis (CF) patients has been associated with a more rapid decline in lung function, increased hospitalisation and mortality. The aim of this study was to evaluate the clonal relationships among 116 MRSA isolates from 12 chronically colonised CF pediatric patients over a 6-year period in a Rio de Janeiro CF specialist centre. Isolates were characterised by antimicrobial resistance, SCCmec type, presence of Panton-Valentine Leukocidin (PVL) genes and grouped according to DNA macrorestriction profile by pulsed-field gel electrophoresis (PFGE) and spa gene type. High resistance rates were detected for erythromycin (78%) and ciprofloxacin (50%) and SCCmec IV was the most common type (72.4%). Only 8.6% of isolates were PVL positive. High genetic diversity was evident by PFGE (39 pulsotypes) and of nine that were identified spa types, t002 (53.1%) and t539 (14.8%) were the most prevalent. We conclude that the observed homogeneity of spa types within patients over the study period demonstrates the persistence of such strain lineages throughout the course of chronic lung infection.