Serotype M1 group A Streptococcus, the most common cause of invasive disease in many case series, generally have resisted extensive molecular subtyping by standard techniques (eg, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis). We used automated sequencing of the sic gene encoding streptococcal inhibitor of complement and of a region of the chromosome with direct repeat sequences to unambiguously differentiate 30 M1 isolates recovered from 28 patients in Texas with invasive disease episodes temporally clustered and thought to represent an outbreak. Sequencing of the emm gene was less useful for M1 strain differentiation, and restriction fragment length polymorphism analysis with IS1548 or IS1562 as Southern hybridization probes did not provide epidemiologically useful subtyping information. Sequence polymorphism in the direct repeat region of the chromosome and IS1548 profiling data support the hypothesis that M1 organisms have two main evolutionary lineages marked by the presence or absence of the speA2 allele encoding streptococcal pyrogenic exotoxin A2.