Many human pathogens produce phenotypic variants as a means to circumvent the host immune system and enhance survival and, as a potential consequence, exhibit increased virulence. For example, it has been known for almost 90 y that clinical isolates of the human bacterial pathogen group A streptococci (GAS) have extensive phenotypic heterogeneity linked to variation in virulence. However, the complete underlying molecular mechanism(s) have not been defined. Expression microarray analysis of nine clinical isolates identified two fundamentally different transcriptomes, designated pharyngeal transcriptome profile (PTP) and invasive transcriptome profile (ITP). PTP and ITP GAS differed in approximately 10% of the transcriptome, including at least 23 proven or putative virulence factor genes. ITP organisms were recovered from skin lesions of mice infected subcutaneously with PTP GAS and were significantly more able to survive phagocytosis and killing by human polymorphonuclear leukocytes. Complete genome resequencing of a mouse-derived ITP GAS revealed that the organism differed from its precursor by only a 7-bp frameshift mutation in the gene (covS) encoding the sensor kinase component of a two-component signal transduction system implicated in virulence. Genetic complementation, and sequence analysis of covR/S in 42 GAS isolates confirmed the central role of covR/S in transcriptome, exoproteome, and virulence modulation. Genome-wide analysis provides a heretofore unattained understanding of phenotypic variation and disease specificity in microbial pathogens, resulting in new avenues for vaccine and therapeutics research.