An unusual emm53, T-28/T-non-typeable, iMLS(B) phenotype clone represented a substantial proportion (28.6%) of invasive erythromycin-resistant group A streptococcus (GAS) isolates in the Czech Republic during 2003. Clonal analysis of emm53 isolates between 2001 and 2004 revealed four pulsed-field gel electrophoresis (PFGE) patterns and two emm subtypes. Isolates produced identical PFGE patterns regardless of their invasiveness and/or tetracycline resistance. Multilocus sequence typing classified all isolates as ST340. An ST5 (emm83) isolate, a potential ancestor of ST340, was isolated in the Czech Republic from an impetigo patient in 1988. The Czech emm53/ST340 isolates shared only three of seven alleles with the original Lancefield emm53/ST11 isolate.