Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis.