The appropriate clinical applications of pneumococcal polysaccharide vaccines against recent increases in antimicrobial resistant Streptococcus pneumoniae (S. pneumoniae) urgently require accurate analytical methodologies for determining and characterizing the serotypes. The results of current immunological determinations of serotypes with anti-capsular polysaccharide-specific sera are difficult to interpret in terms of quellung changes of the pneumococci. In this study, we applied the multiplex PCR technique for the rapid identification of pneumococci and simultaneous rapid determinations of their serotypes and genotypes that directly correlated with antimicrobial susceptibilities from nasopharyngeal secretions (NPS). Serogroups 6, 19F and 23F were the predominant capsular types of S. pnuemoniae in the NPS samples. Strains of serotypes 19F and 23F frequently had mutations in pbp1a, pbp2x and pbp2b and expressed ermB and mefA; they also were mostly resistant to both penicillin G (PCG) and clarithromycin (CAM). Two NPS samples contained the strain of serotype 19F together with the strain of serotype 23F, although only the strain of serotype 19F was identified by a conventional bacterial culture. Pneumococci were identified in six NPS samples and their serotypes determined by the multiplex PCR, while a conventional bacterial culture failed to identify the pathogens. Our findings suggest that PCR-based serotyping and genotyping can provide an accurate and rapid distribution of pneumococcal serotypes and antimicrobial resistance. The relatively minor populations in the nasopharynx may be determined using molecular techniques.