To better understand the molecular events involved in the origin of new pathogenic bacteria, we studied the evolution of a highly virulent clone of serotype M1 group A Streptococcus (GAS). Genomic, DNA-DNA microarray, and single-nucleotide polymorphism analyses indicated that this clone evolved through a series of horizontal gene transfer events that involved (1) the acquisition of prophages encoding streptococcal pyrogenic exotoxin A and extracellular DNases and (2) the reciprocal recombination of a 36-kb chromosomal region encoding the extracellular toxins NAD⁺-glycohydrolase (NADase) and streptolysin O (SLO). These gene transfer events were associated with significantly increased production of SLO and NADase. Virtual identity in the 36-kb region present in contemporary serotype M1 and M12 isolates suggests that a serotype M12 strain served as the donor of this region. Multiple horizontal gene transfer events were a crucial factor in the evolutionary origin and emergence of a very abundant contemporary clone of serotype M1 GAS