Objective To analyze the molecular biological characteristics of 13 standard strains of Streptococcus pneumoniae preserved by the National Center for Medical Culture Collections (CMCC). Methods Thirteen standard strains of Streptococcus pneumoniae were first identified by the traditional culture method, microscopic observation, serum agglutination test and mass spectrometry, and followed by the 16S rRNA gene analysis and multilocus sequence type (MLST). Results The results of traditional culture, microscopic observation, serum agglutination and mass spectrometry of 13 standard strains were consistent with the characteristics of Streptococcus pneumoniae. The expected size of 16S rRNA gene fragment was amplified from all the 13 standard strains, and its similarity with the 16S rRNA gene sequence (LN831051) of Streptococcus pneumoniae type strain NCTC 7465 was more than 99.00%. MLST results showed that the 13 standard strains could be divided into 13 sequence types (STs), which were ST2296, ST180, ST2759, ST3844, ST12973, ST12977, ST3545, ST16632, ST875, ST10189, ST870, ST342 and ST7234, respectively. The ST16632 of strain CMCC(B)31547 was a newly discovered ST type. Conclusion The 16S rRNA gene sequences and MLST typing data of 13 different serotypes of Streptococcus pneumoniae standard strains are obtained, which provides the data support for the traceability and further improvement of quality standards of Streptococcus pneumoniae.