Polymerase chain reaction (PCR) analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis was useful for identification of staphylococci and for strain delineation of Staphylococcus aureus. In the study presented in this chapter, 40 ITS patterns were demonstrated among 228 isolated colonies of S. aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA); 11 patterns for methicillin-resistant S. aureus (MRSA); and 3 patterns for both MSSA and MRSA, highlighting the inability of ITS pattern analysis to differentiate the MSSA and MRSA strains. To overcome this problem, simultaneous PCR amplification of the ITS region and the mecA gene was applied to isolated colonies of staphylococcus species and positive-testing blood culture bottles.