Objective: To understand the homology and resistance characteristics of Streptococcus pneumoniae (S. pneumoniae) in healthy preschool children, so as to provide basis for disease transmission prevention and rational use of antibiotics. Methods: Stratified cluster random sampling method was used to sample 1 829 healthy children from six kindergartens in Shunde District, Foshan City. Nasal swabs were taken and tested for S. pneumoniae. Multi locus sequence typing was used for homology analysis. The Chi squared test and random forest analysis were used to explore the resistance characteristics. Results: The nasal carriage rate of S. pneumoniae and multidrug resistant S. pneumoniae (MDRSP) in children were 22.5%(412/1 829) and 21.3%(390/1 829), respectively. Homology analysis in sequence types showed that the total homology rates of 6 kindergartens were 93.5%(87/93), 91.1% (72/79), 89.2%(58/65), 88.9%(64/72), 86.2%(50/58), 77.8%(35/45), respectively. It was found that the highest homology rate was 82.8% (48/58) within class and 93.1% (81/87) between classes. S. pneumoniae was mainly resistant to azithromycin (97.1%, 400/412), erythromycin (92.0%, 379/412) and tetracycline (91.5%, 377/412). The dominant multidrug resistance pattern of MDRSP isolates was not sensitive to azithromycin, erythromycin, cotrimoxazole, tetracycline and clindamycin. Random forest analysis indicated that the important phenotypic markers associated with MDRSP were resistance to azithromycin, cotrimoxazole, tetracycline, clindamycin and erythromycin(MDG=8.94, 6.92, 5.80, 4.84, 2.58). Conclusion The risk of cross transmission of S. pneumoniae among preschool children is high, and direct contact is the main way of transmission. Consequently, kindergartens and health departments should take effective measures to effectively prevent and block the spread of Streptococcus pneumoniae.