The main purpose of the research is to evaluate the application feasibility of multiplex polymerase chain reaction (PCR) in serotyping of Streptococcus pneumoniae. A total of 568 strains were subjected to multiplex PCR and capsular swelling test respectively. The results demonstrated that 37.5% of the strains (213/568) could be successfully serotyped by capsular swelling test into 16 different serotypes, including serotypes 19 (23.1%, 131/568), 6 (5.3%, 30/568), 23 (1.6%, 9/568), 14 (1.4%, 8/568), 9 (1.1%, 6/568) and 15 (1.1%, 6/568); while 62.7% of the strains (356/568) could be successfully serotyped by multiplex PCR into 21 different serotypes, including serotypes 19 (27.8%, 158/568), 23 (8.5%, 48/568), 6 (7.4%, 42/568), 14 (4.4%, 25/568), 3 (4.2%, 24/568) and 15 (3.5%, 20/568). The multiplex PCR could not identify serotypes 4 and 18 which could be identified by capsular swelling test, while the capsular swelling test could not identify serotypes 5, 12, 35, 16, 17 and 22 which could be successfully identified by multiplex PCR. There was no significant difference between the two methods to identify serotypes 19F and 19A. The results suggest that there is significant difference between the two methods in serotyping of Streptococcus pneumoniae. Multiplex PCR is more effective than the conventional capsular swelling test, especially for those specimens collected from complicated sources. The combination of the two methods can improve the serotyping efficiency of Streptococcus pneumoniae.