Group B streptococcus (GBS; Streptococcus agalactiae) is a leading cause of neonatal invasive infections, and until recently, it was thought to be completely susceptible to penicillin. However, we recently identified several clinical GBS isolates with reduced penicillin susceptibility (PRGBS) whose minimum inhibitory concentrations of penicillin were >0.12 μg/ml, which is above the susceptibility breakpoint set by the Clinical and Laboratory Standards Institute. These PRGBS were isolated between 1995 and 2005 in Japan; whether these PRGBS existed in Japan before 1995 is unknown. In the study described here, we screened for PRGBS among 349 clinical GBS isolates obtained in Japan between 1977 and 2005 using the previously developed disk diffusion method for the detection of PRGBS. With this method, we selected 6 PRGBS candidates and confirmed that 1 isolate was PRGBS, using agar dilution method, including oxacillin, ceftizoxime, and penicillin-binding protein 2X (PBP2X) gene sequencing analysis. This isolate was obtained from sputum in 2005, and we could not detect PRGBS isolates before 1995 in this investigation.