We have developed a rapid and simplified approach for the strain characterization of Staphylococcus aureus on the basis of multilocus sequence typing (MLST) in which sequence variations in the MLST housekeeping gene loci are detected by restriction fragment pattern analysis rather than sequencing; we refer to this approach as multilocus restriction fragment typing (MLRFT). Briefly, MLRFT for S. aureus involves the PCR amplification of each of the seven MLST housekeeping gene loci by using the same primer pairs used in MLST. The amplicons are then digested directly with one or two restriction enzymes and the restriction fragments are resolved by agarose gel electrophoresis. Projection from published MLST data shows that MLRFT captures about 95% of the genetic diversity detected by MLST. The MLRFT approach was validated with a set of 59 methicillin-susceptible and 44 methicillin-resistant S. aureus isolates from community-acquired and nosocomial sources which had previously been characterized by pulsed-field gel electrophoresis (PFGE). MLRFT resolved the 103 isolates into 15 restriction fragment types, giving a discrimination index of 89.0%. Clonal groupings established by MLRFT correlated well with those established by PFGE. In short, MLRFT provides a convenient alternative to MLST and PFGE because it requires minimal laboratory facilities and is relatively simple and inexpensive to perform.