Group A streptococcus (Strep A) surface M protein, an α-helical coiled-coil dimer, is a vaccine target and a major determinant of streptococcal virulence. The sequence-variable N-terminal region of the M protein defines the M type and also contains epitopes that promote opsonophagocytic killing of streptococci. Recent reports have reported considerable cross-reactivity among different M types, suggesting the prospect of identifying cross-protective epitopes that would constitute a broadly protective multivalent vaccine against Strep A isolates. Here, we have used a combination of immunological assays, structural biology and cheminformatics to construct a recombinant M protein-based vaccine that included six Strep A M peptides that were predicted to elicit antisera that would cross-react with an additional 15 non-vaccine M types of Strep A. Rabbit antisera against this recombinant vaccine cross-reacted with 10 of the 15 non-vaccine M peptides. Two of the five non-vaccine M peptides that did not cross-react shared high sequence identity (≥ 50%) with the vaccine peptides, implying that high sequence identity alone was insufficient for cross-reactivity among the M peptides. Additional structural analyses revealed that the sequence identity at corresponding polar helical-wheel heptad sites between vaccine and non-vaccine peptides accurately distinguishes cross-reactive from non-cross-reactive peptides. On the basis of these observations, we developed a scoring algorithm based on the sequence identity at polar heptad sites. When applied to all epidemiologically important M types, this algorithm should enable the selection of a minimal number of M peptide-based vaccine candidates that elicit broadly protective immunity against Strep A.,Published under license by The American Society for Biochemistry and Molecular Biology, Inc.