OBJECTIVE To introduce a novel molecular PCR method for the typing of Streptococcus pneumonia in the National Reference Laboratory (NRL) for Streptococcal Infections. METHODS Strains of Streptococcus pneumoniae are referred to the NRL from different regions of the Czech Republic. Generally, the identification and typing are based on strain morphology, optochin susceptibility, bile solubility, latex agglutination, and the Quellung reaction. Since 2012, a novel multiplex polymerase chain reaction (mPCR) assay has been introduced. The novel assay was tested on 210 S. pneumoniae isolates and 8 isolates of the related species S. pseudopneumoniae, S. sanguinis, and S. oralis. RESULTS The NRL for Streptococcal Infections has included a novel mPCR assay in the algorithm of S. pneumoniae identification and typing. The mPCR assay was able to identify and type any pneumococcal strain from the study collection, with the isolates of the related species remaining negative. The mPCR assay showed 100% sensitivity and specificity in this study. The pCR appeared to be an excellent tool for S. pneumoniae typing. CONCLUSIONS Until recently, S. pneumoniae serotypes and serogroups were differentiated using a serological approach (Quellung reaction), but the NRL for Streptococcal Infections has switched to a novel mPCR assay. This molecular tool improves S. pneumoniae typing, making it more accurate.