Multilocus sequence typing (MLST) is a tool that can be used to study the molecular epidemiology and population genetic structure of microorganisms. A MLST scheme was developed for Streptococcus pyogenes and the nucleotide sequences of internal fragments of seven selected housekeeping loci were obtained for 212 isolates. A total of 100 unique combinations of housekeeping alleles (allelic profiles) were identified. The MLST scheme was highly concordant with several other typing methods. The emm type, corresponding to a locus that is subject to host immune selection, was determined for each isolate; of the >150 distinct emm types identified to date, 78 are represented in this report. For a given emm type, the majority of isolates shared five or more of the seven housekeeping alleles. Stable associations between emm type and MLST were documented by comparing isolates obtained decades apart and/or from different continents. For the 33 emm types for which more than one isolate was examined, only five emm types were present on widely divergent backgrounds, differing at four or more of the housekeeping loci. The findings indicate that the majority of emm types examined define clones or clonal complexes. In addition, an MLST database is made accessible to investigators who seek to characterize other isolates of this species via the internet (http://www.mlst.net).