Streptococcus Pneumoniae is a leading cause of acute otitis media (AOM). For most AOM caused by S. pneumoniae, penicillin is the antibiotic of choice. However, there are some recent reports of clinical resistance to penicillin by S. pneumoniae. The sequences of penicillin binding protein, pbpla, pbp2b and pbp2x, genes of penicillin-resistant S. pneumoniae (PRSP) were more highly divergent than those of penicillin-succeptible S. pneumoniae (PSSP). The polymerase chain reaction (PCR) can easily determine whether an S. pheumoniae isolate is susceptible or resistant to penicillin by amplifying the target gene by using a combination of primers. In this study, clinical isolates (n = 12) were obtained from the nasopharynx of patients with AOM. PCR was used to confirm the identification of an isolate as S. pneumoniae by amplifying the autolysin gene and to detect three PBP genes by amplifying parts of pbp1a, pbp2x and pbp2b. The resistance of S. pneumoniae to penicillin and other beta-lactams has been shown to be associated with mosaic mutations in the pbp1a, pbp2b and pbp2x genes. These findings suggest that rapid identification of PSSP and PISP/PRSP by PCR is possible and very useful for proper treatment of acute otitis media.