Clinical relevance of multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in patients with community-acquired pneumonia (CAP) by Mycoplasma pneumoniae (M. pneumoniae) is unknown. A multi-center, prospective study was conducted from November 2010 to April 2012. Nine hundred and fifty-four CAP patients were consecutively enrolled. M. pneumoniae clinical isolates were obtained from throat swabs. MLVA typing was applied to all isolates. Comparison of pneumonia severity index (PSI) and clinical features among patients infected with different MLVA types of M. pneumoniae were conducted. One hundred and thirty-six patients were positive with M. pneumoniae culture. The clinical isolates were clustered into 18 MLVA types. One hundred and fourteen (88.3%) isolates were resistant to macrolide, covering major MLVA types. The macrolide non-resistant rate of M. pneumoniae isolates with Mpn13-14-15-16 profile of 3-5-6-2 was significantly higher than that of other types (p ≤ 0.001). Patients infected with types U (5-4-5-7-2) and J (3-4-5-7-2) had significantly higher PSI scores (p<0.001) and longer total duration of cough (p = 0.011). Therefore it seems that there is a correlation between certain MLVA types and clinical severity of disease and the presence of macrolide resistance.