Bacteria of the Burkholderia cepacia complex consist of a number of closely related genomic species (genomovars) potentially pathogenic for cystic fibrosis (CF) patients, collectively referred to as the B. cepacia complex. The genomovar status and epidemiological relatedness of B. cepacia complex strains recovered from CF patients, attending a CF Center at the University Hospital "Policlinico Umberto I" of Rome, were investigated using 16S rRNA PCR-RFLP, recA PCR-RFLP, genomovar-specific PCR, and RAPD. Forty-seven isolates identified as B. cepacia by commercial systems were repeatedly recovered from 19 CF patients. The taxonomy approach used in this study showed that 17 of the 19 patients were colonized by B. cepacia complex strains. Genomovar III (11 strains) was the most prevalent genomovar. Two strains of genomovar I, one B. stabilis (genomovar IV), one B. multivorans (genomovar II), and 4 strains of B. anthina (genomovar VIII) were also identified. This is the first report of multiple patient colonization by B. anthina in a CF center. The epidemiological and genetic relatedness as well as the presence of molecular markers associated with virulence and transmissibility of the B. cepacia complex strains were determined and probable patient-to-patient spread was observed.