The depth of knowledge concerning its physiology and genetics make Bacillus subtilis an attractive system for strain engineering and analysis. Transposon-based mutagenesis strategies generate large libraries of mutant strains that can be used to investigate molecular mechanisms relevant in fundamental research or to generate desirable phenotypes in applied research. This section presents a mini-Tn10-based transposon mutagenesis system that is capable of genome-wide insertional mutagenesis in B. subtilis and related organisms. Using appropriately designed selections or screens, the desired strain phenotypes can be isolated from transposon mutant libraries. This transposon system then allows rapid identification of the genetic locus responsible for the desired phenotype, and, due to the natural competence of B. subtilis, the identified genotypic change can easily be confirmed as responsible for the phenotypic change.