Polymerase chain reaction (PCR) identification assays were designed for eight major species of coagulase-negative staphylococci (CNS) on the basis of three variable regions found in the 16S rRNA gene. The PCR assays were tested with 41 staphylococcal strains representing the diversity of staphylococci defined by classical biotyping schemes. Each PCR result was compared with species-specific polymorphism in and around the 16S rRNA gene (i.e., 16S ribotype) and the phenotypic identification of the strain in a miniaturised biochemical test gallery (bioMerieux ATB 32 Staph). Twenty-six of the 41 strains were identified by PCR as belonging to one of the eight species for which primers had been designed and none of the remaining strains was misidentified. For 22 of the 26 strains there was complete agreement between the PCR identification, 16S ribotype and ATB identification. For the remaining four strains there was agreement between PCR identification and 16S ribotype. Two National Collection of Type Culture strains were re-assigned to different species and 10 previously unassigned strains were formally speciated for the first time. These PCR assays are suitable for rapid and definitive speciation of CNS.