Strains with 93–95 % gyrB gene sequence similarity exhibited DNA–DNA relatedness of 60–70 % without exception, indicating that they are grouped at the subspecies level. Exceptionally, B. subtilis subsp. spizizenii BCRC 17366T and B. vallismortis BCRC 17183T showed 93.9 % gyrB gene sequence similarity and 52 % DNA–DNA relatedness. This finding indicated that it might be necessary to use several gene sequences and DNA–DNA hybridization to discriminate species relationships. Nevertheless, based on data obtained from the present study, the gyrB gene sequences have been shown to be a more efficient phylogenetic tool than the 16S RNA gene sequences for discriminating between species of this group.