Recent taxonomic advances have demonstrated that Burkholderia cepacia is a cluster of at least seven closely related genomic species (or genomovars) collectively referred to as the B. cepacia complex, all of which may cause infections among cystic fibrosis patients and other vulnerable individuals. Thus, it is important for clinical microbiologists to be able to differentiate genomovars. Prior to this study, 361 B. cepacia complex isolates and 51 isolates easily confused with B. cepacia complex previously had been identified using a polyphasic approach, and in this study, a comparison of phenotypic and biochemical tests was carried out. It was determined that Burkholderia multivorans and Burkholderia stabilis could reliably be separated from other members of the B. cepacia complex by phenotypic methods. A combination of phenotypic and molecular tests such as recA PCR and 16S rRNA RFLP are recommended for differentiation among the genomovars of the B. cepacia complex. A biochemical reaction scheme for the identification of B. gladioli, Pandoraea species, and Ralstonia pickettii and the differentiation of these species from the B. cepacia complex is also presented.