Objectives: To assess the macrolide resistance, phenotype, and genotypic characterization of Streptococcus pyogenes isolated from Chinese children with pharyngitis. Methods: Minimal inhibitory concentration (MIC) with nine antibiotics was determined on 188 isolates of S. pyogenes collected from outpatients with pharyngitis in four children's hospitals in different regions of China in 2007. MICs of penicillin, chloramphenicol, cefradine, levofloxacin, macrolide (erythromycin, clarithromycin, azithromycin), clindamycin, and tetracycline were determined by the microdilution method. The macrolide resistant phenotypes of isolates were determined through a double-disk. The macrolide-resistant genes (mefA, ermB, and ermA) were amplified by polymerase chain reaction (PCR). Results: Over 95% were resistant to macrolides, while 92.0% were resistant to tetracycline. We also found that all isolates were sensitive to penicillin, chloramphenicol, cefradine, and levofloxacin. Among the 173 erythromycin resistant strains, 171 (98.8%) were assigned to the cMLS phenotype, while the remaining 2 (1.2%) were assigned to the iMLS phenotype. Among the 171 cMLS isolates, 168 isolates (98.2%) had the ermB gene accounting for 98.2%. Meanwhile, 2 iMLS isolates had the ermA gene. Macrolides were highly resistant to ermB positive strains (MIC90 > 256 mu g/ml). Neither the M-phenotype nor the mefA gene was detected. Meanwhile, our studies of multiple centers showed that consumption of macrolides from 2000 to 2006 was very high. Conclusion: The main phenotype is cMLS, and the ermB gene code is the main resistance mechanism against macrolides in S. pyogenes. The high rate of macrolide resistance to S. pyogenes was observed, which may be correlated with the overuse of these antibiotics in China. Pediatr Pulmonol. 2009; 44:436-441. (C) 2009 Wiley-Liss, Inc.