Fifty-three Mycoplasma pneumoniae strains were isolated from pediatric patients in Shanghai, China, from October 2005 to February 2008. Of 53 clinical isolates, 44 (83%) were resistant to erythromycin (MICs of >128 microg/ml for all 44 strains), azithromycin, and clarithromycin. All macrolide-resistant M. pneumoniae strains harbored an A-to-G transition mutation at position 2063 in 23S rRNA genes. Forty-five (85%) clinical isolates were classified into the P1 gene restriction fragment length polymorphism type I, and six (11%) were type II.