We evaluated the use of a novel multiple-locus variable-number tandem-repeat analysis (MLVA) method for typing of human Staphylococcus aureus. For a total of 150 clinical isolates, MLVA demonstrated the highest discriminatory power. MLVA correctly assigned isolates to outbreaks or identified isolates as unlinked. MLVA is a rapid and simple method for the epidemiological typing of S. aureus.