CsrS/CsrR is a 2-component system in Streptococcus pyogenes that negatively regulates hyaluronic capsule and several exotoxins. To detect spontaneous mutations in csrRS mucoid and large colony variants of M1 strain MGAS166 were isolated from experimental murine skin infections. By use of complementation with a csrRS⁺ plasmid, relevant mutations were also detected in 7 of 12 human clinical isolates. The presence of spontaneous mutants in mouse infection was associated with larger, more necrotic lesions. Most spontaneous changes in CsrR resulted from single amino acid substitutions, whereas most csrS mutations were frameshift or nonsense mutations. In 2 instances, IS1548 insertions were found in csrS. Experimental inoculation of mixtures of wild-type (wt) and csrRS⁻ bacteria yielded larger, more necrotic lesions than did either strain at twice the inoculum, which suggests that these variants may exhibit pathogenic synergy. Spontaneous emergence of csrRS⁻ mutants in vivo enhances the virulence of wt bacteria and increases severity of murine skin infection