ABSTRACT We developed a multiplex PCR-based methodology for nasopharyngeal samples maintained in egg thioglycolate antibiotic and skim milk-tryptone-glucose-glycerol media to identify and serotype the most important serotypes of Streptococcus pneumoniae that cause invasive disease in children. This technique can be used to study the epidemiology of pneumococcal colonization and the effect of conjugate vaccines.