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Objective To investigate drug resistance features and homology among penicillin-intermediate Streptococcus pneumoniae isolates from Wenzhou City, China. Methods Fifty-one penicillin-intermediate S. pneumoniae isolates were obtained from respiratory samples of infants and children hospitalized with lung infections. An antimicrobial susceptibility test was used to assess drug resistance. Polymerase chain reaction and agarose gel electrophoresis were used to identify S. pneumoniae isolates and pulsed-field gel electrophoresis (PFGE) was used to analyze molecular subtypes. Hierarchical cluster analysis of PFGE fingerprints was used to compare genetic diversity and relatedness of S. pneumoniae isolates. The Quellung test was used for serotyping. Results Fifty-one penicillin-intermediate S. pneumoniae isolates showed evidence of multi-drug resistance and polyclonal origins. The isolates were classified into 25 subtypes through hierarchical cluster analysis of PFGE fingerprints. Three of these subtypes formed a supertype (15/51, 16/51 and 8/51 isolates), while the remaining subtypes occurred sporadically (12/51 isolates). Conclusions Transmission of penicillin-intermediate S. pneumoniae is mostly vertical and to a lesser extent horizontal. Effective prevention strategies, including respiratory tract management and contact isolation, are essential to control nosocomial S. pneumoniae infection. Once susceptibility is confirmed, vancomycin, high-dose penicillin or third-generation cephalosporins (cefotaxime and ceftriaxone) may be used to treat penicillin-intermediate S. pneumoniae.