Group B streptococci (GBS) are pathogens of both neonates and adults, with serotype III strains in particular being associated with invasive disease and meningitis. In this study, a novel GBS surface antigen, ε, was found to be co-expressed with the previously reported δ antigen on an identical subset of serotype III GBS. Expression of δ/ε on the surface of serotype III GBS was shown to distinguish the restriction digest pattern (RDP) III-3 and multilocus sequence typing (ST)-17 lineage. ε-Specific antibodies were reactive with a unique, high-molecular-mass, serine-rich repeat protein (Srr-2) found exclusively in RDP III-3 strains. The gene encoding Srr-2 was located within a putative accessory secretory locus that included secY2 and secA2 homologues and had a genetic organization similar to that of the secY2/A2 locus of staphylococci. In contrast, serotype III δ/ε-negative strains and strains representative of serotypes Ia, Ib, Ic and II shared a common Srr-encoding gene, srr-1, and an organization of the secY2/A2 locus similar to that of previously reported serotype Ic, δ/ε-negative serotype III and serotype V GBS strains. Representative serotype III δ/ε-positive strains had LD₉₀ values 3–4 logs less than those of serotype III δ/ε-negative strains in a neonatal mouse model of infection. These results indicate that the RDP III-3/ST-17 lineage expresses Srr-2 and is highly virulent in an in vivo model of neonatal sepsis.