No data on the genetic analysis of penicillin-resistant Streptococcus pneumoniae (PRP) in Japan has been reported. The SmaI restriction endonuclease digested patterns of chromosomal DNAs from 15 PRP serotyped 23F isolated in Japan were analyzed by pulsed-field gel electrophoresis (PFGE). The isolates were genetically heterogeneous and seven different PFGE patterns were identified. Nine strains were also resistant to erythromycin and tetracycline. Four strains revealed resistance to ceftriaxone. The PFGE patterns of some strains isolated in Nagasaki University Hospital were identical to each other and closely resembled those of isolates from three different areas of Japan. These results indicate a need for additional studies by PFGE to determine the possibility of clonal spread in Japan.