The aim of this study was to describe the genetic characteristics of Streptococcus pyogenes showing the MLSB phenotype of macrolide resistance from 1999 to 2005 in Spain and to highlight the substantial increase in these isolates in the last few years. The antimicrobial susceptibilities of 17,232 group A streptococci isolated from Madrid and Gipuzkoa from 1999 to 2005 were studied. The presence of the resistance genes ermA, ermB, mef, tetM, and tetO and the presence of the intTn and xis genes of the Tn916-Tn1545 transposon family were studied in a sample of 739 MLSB-resistant isolates. The epidemiological relationships among these isolates were analyzed by emm typing, T typing, and multilocus sequence typing. Erythromycin resistance was found in 21.3% of the isolates analyzed (annual variation of 14.3% to 28.9%). Until 2003, most erythromycin-resistant isolates showed the M phenotype, but in 2004 and 2005, about 50% of isolates showed the MLSB phenotype. Among the MLSB-resistant isolates studied, 16 clones were identified. The most prevalent clone was a strange emm11/T11/ST403 clone with a null yqiL allele. All but one of the 463 emm11/T11/ST403 isolates carried the ermB, tetM, intTn, and xis genes. The second most prevalent MLSB-resistant clone was emm28/T28/ST52, which comprised two subclones: one bacitracin-resistant, tetracycline-susceptible subclone carrying the ermB gene (n=115) and another bacitracin-susceptible, tetracycline-resistant subclone carrying the ermB and tetM genes (n=33). The rapid diffusion of these two clones, and especially of emm11/T11/ST403, caused the large increase in MLSB-resistant S. pyogenes isolates in Spain, suggesting a potential ability for international dissemination.