Objective The aim of this study is to investigate the macrolide resistance rate and molecular type withmultiple-locus variable-number tandem-repeat analysis (MLVA) of Mycoplasma pneumoniae of Beijing in 2016 in pediatric patients. Methods Real-time quantitative polymerase chain reaction (PCR) was used to identify M. pneumoniae, and MLVA was performed. The domain V of the 23S rRNA was sequenced to detect macrolide-resistant point mutations. We also investigated the activities of antibiotics against M. pneumoniae isolates in vitro. Results The PCR detection rate of M. pneumoniae in children in Beijing was 40％, and the macrolide resistance rate was 66％. The A2063G mutation in the 23S rRNA V region is the dominant mutation (137/146, 93.84％), whereas the A2064G mutation is rare (9/146, 6.16％). Seventy-three samples were typed successfully by MLVA typing, including 86.3％ (63/73) were MLVA type 4-5-7-2, and 13.7％ (10/73) were MLVA type 3-5-6-2. No other types were found. No strains were resistant to levofloxacin or tetracycline. Conclusion In 2016, a specific decrease in the macrolide resistance rate occurred in Beijing. The detection rate and macrolide resistance rate of outpatients are lower than those of inpatients. The A2063G mutants M. pneumoniae have high levels of resistance to erythromycin and azithromycin. The primary MLVA type is 4-5-7-2, followed by 3-5-6-2. No other MLVA types were detected. No strains resistant to tetracycline or levofloxacin were found in vitro.