Given the widespread presence of Burkholderia cenocepacia in the rhizosphere it is important to determine whether rhizosphere strains are pathogenic for cystic fibrosis patients or not. Eighteen B. cenocepacia strains of rhizosphere and clinical origin were typed by multi-locus sequence typing (MLST) analysis and compared for their ability to invade pulmonary epithelial cells and their virulence in a mouse model of airway infection. Although there was great variability, clinical strains were the most invasive in vitro. Almost all the rhizosphere and two clinical strains were defined as non-invasive, six clinical strains as invasive, and two strains of both clinical and environmental origin as indeterminate. Exposure of murine airways to clinical strains caused higher acute mortality than that seen after challenge with rhizosphere strains. Furthermore, both clinical and environmental strains were able to persist in the lungs of infected mice, with no significant differences in bacterial loads and localization 14 days after challenge. DNA dot blot analyses of AHL synthase, porin and amidase genes, which play a role in B. cenocepacia virulence, showed that they were present in B. cenocepacia strains irrespective of their origin. Overall, our results suggest that rhizosphere strains do not differ from clinical strains in some pathogenic traits.