The taxonomic position of five recA gene clusters of Burkholderia cepacia complex (Bcc) isolates was determined using a polyphasic taxonomic approach. The levels of 16S rRNA and recA gene sequence similarity, multilocus sequence typing (MLST) data and the intermediate DNA–DNA binding values demonstrated that these five clusters represented five novel species within the Bcc. Biochemical identification of these species is difficult, as is the case for most Bcc species. However, identification of these novel species can be accomplished through recA gene sequence analysis, MLST and BOX-PCR profiling and by recA RFLP analysis. For diagnostic laboratories, recA gene sequence analysis offers the best combination of accuracy and simplicity. Based on these results, we propose five novel Bcc species, Burkholderia latens sp. nov. (type strain FIRENZE 3^T =LMG 24064^T =CCUG 54555^T), Burkholderia diffusa sp. nov. (type strain AU1075^T =LMG 24065^T =CCUG 54558^T), Burkholderia arboris sp. nov. (type strain ES0263A^T =LMG 24066^T =CCUG 54561^T), Burkholderia seminalis sp. nov. (type strain AU0475^T =LMG 24067^T =CCUG 54564^T) and Burkholderia metallica sp. nov. (type strain AU0553^T =LMG 24068^T =CCUG 54567^T). In the present study, we also demonstrate that Burkholderia ubonensis should be considered a member of the Bcc.