The emerging clinical importance of staphylococcal infections prompted us to establish a reference database for partial RNA polymerase B (rpoB; nucleotides 1444-1928) gene sequences from type strains of all staphylococcal species and subspecies. This database correctly identified 55 clinical staphylococcal isolates; all were correctly identified at the species level. At the subspecies level, rpoB misidentified only 2 isolates.