Background: Although group B Streptococcus (GBS; i.e. Streptococcus agalactiae) has been considered to be uniformly susceptible to β-lactams, GBS isolates with reduced penicillin susceptibility (PRGBS) have been reported from Japan and North America. In this study, PRGBS from Japan were characterized by multilocus sequence typing (MLST) and the results compared with data on PRGBS reported from the USA. Methods: Twenty-eight clinical isolates of PRGBS recovered in Japan (including 22 isolates previously analysed by PFGE) were analysed by MLST and eBURST (http://eburst.mlst.net/). Results: Twenty-three isolates were found to belong to the sequence type 1 (ST1) group (11 ST458, 7 ST1, 3 ST297, 1 ST358 and 1 ST4), while the remaining 5 isolates formed the ST23 group. Among 11 ST458 and 7 ST1 isolates, 9 and 4 were serotype VI, respectively, indicating a probable correlation between the ST1 group and serotype VI for PRGBS in Japan. Conclusions: PRGBS in Japan could be classified into at least two ST groups, ST1 and ST23, which are genetically different from the ST19 PRGBS isolated in the USA, though five allele variations were seen between ST1 and ST19, implying a slight genetic relatedness. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.