Staphylococcus aureus is the leading cause of wound and hospital-acquired infections. The emergence of strains with resistance to all antibiotics has created a serious public health problem. Transposon-based mutagenesis can be used to generate libraries of mutants and to query genomes for factors involved in nonessential pathways, such as virulence and antibiotic resistance. Ideally, such studies should employ defined and complete sets of isogenic mutants and should be conducted so as to permit acquisition and comparison of the complete data sets. Such systematic knowledge can reveal entire pathways and can be exploited for the rational design of therapies. The mariner-based transposon, bursa aurealis, can be used to generate random libraries of mutants in laboratory strains and clinical isolates of S. aureus. This chapter describes a procedure for isolating mutants and mapping the insertion sites on the chromosome.