During 2000-2007 in Lisbon, we identified 45 bacitracin-resistant Streptococcus pyogenes isolates among 1629 isolates: 24 from oropharyngeal healthy carriers (out of 1026), 21 from patients with noninvasive infections (out of 559) and zero from invasive infections (out of 44). Forty-four of those isolates, mainly of colonization, are low-level bacitracin-resistant members of the cMLS(B)-macrolide-resistant and tetracycline-susceptible emm28/ST52 clone previously detected in Europe, but only among clinical samples. One high-level bacitracin-resistant isolate, associated with a tonsillitis/pharyngitis episode, is cMLSB-macrolide-resistant and tetracycline-resistant member of the emm74/ST120 lineage, which was not previously known to include bacitracin-resistant isolates. The bcrABDR operon encoding an ATP-binding cassette transporter in Enterococcus faecalis was not detected among these bacitracin-resistant S. pyogenes strains. Virulence profiling indicated that genes coding for exotoxins and superantigens seem to be clone specific. This study provides an increased knowledge about specific bacitracin-resistant S. pyogenes strains, which may be useful in future investigations aiming to understand the mechanism(s) leading to bacitracin resistance and the cause(s) for differences in colonization and/or dissemination potential.