Streptococcus pyogenes (group A streptococcus) is remarkable in terms of the large number of diseases it can cause in humans and for the large number of streptococcal factors that have been identified as potential virulence determinants for these diseases. A challenge is to link the function of potential virulence factors to the pathogenesis of specific diseases. An exciting advance has been the development of sophisticated genetic systems for the construction of loss-of-function, conditional, hypomorphic, and gain-of-function mutations in targeted S. pyogenes genes that can be used to test specific hypotheses regarding these genes in pathogenesis. This will facilitate a mechanistic understanding of how a specific gene function contributes to the pathogenesis of each streptococcal disease. Since the first S. pyogenes genome was completed in 2001, hundreds of complete and draft genome sequences have been deposited. We now know that the average S. pyogenes genome is approximately 1.85 Mb and encodes ∼1,800 genes and that the function of most of those genes in pathogenesis remains to be elucidated. However, advances in the development of a variety of genetic tools for manipulation of the S. pyogenes genome now provide a platform for the interrogation of gene/phenotype relationships for individual S. pyogenes diseases, which may lead to the development of more sophisticated and targeted therapeutic interventions. This article presents an overview of these genetic tools, including the methods of genetic modification and their applications.