PURPOSE: We aimed to establish the prevalence of different Burkholderia species among UK cystic fibrosis (CF) and non-CF patients over a 2 year period. METHODOLOGY: Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to identify isolates to genus level, followed by recA/gyrB sequence clustering or species-specific PCR. In all, 1047 Burkholderia isolates were submitted for identification from 361 CF patients and 112 non-CF patients, 25 from the hospital environment and three from a commercial company. Potential cross-infection was assessed by pulsed-field gel electrophoresis (PFGE) and multi- locus-sequence typing (MLST). MICs were determined for 161 Burkholderia cepacia complex (Bcc) isolates. CF Trust registry data were sought to examine clinical parameters relating to Bcc infection. RESULTS: Burkholderia multivorans was the most prevalent species among CF patients affecting 56 % (192) patients, followed by Burkholderia cenocepacia IIIA (15 %; 52 patients). Five novel recA clusters were found. Among non-CF patients, Burkholderia cepacia was the most prevalent species (37/112; 34 %), with 18 of 40 isolates part of a UK-wide B. cepacia 'cluster'. This and three other clusters were investigated by PFGE and MLST. Cable-pili positive isolates included two novel sequence types and representatives of ET12. Antibiotic susceptibility varied between and within species and CF/non- CF isolates. CF Trust registry data suggested no significant difference in lung function between patients harbouring B. cenocepacia, B. multivorans and other Bcc species (P=0.81). CONCLUSION: The dominance of B. multivorans in CF, the presence of a B. cepacia cluster among non-CF patients and the existence of putative novel species all highlighted the continuing role of Burkholderia species as opportunistic pathogens.