We studied a collection of 110 serotype III group B streptococcus (GBS) isolates causing neonatal meningitis, by means of both pulsed-field gel electrophoresis (PFGE) with SmaI and Southern hybridization with probes for genes potentially associated with virulence (neuA, cpsA, scpB, and hylB and, for mobile genetic elements [MGEs], GBSi1 and IS1548), in comparison with 44 serotype III GBS isolates colonizing healthy neonates. Using polymerase chain reaction, we assessed both the insertion of MGEs downstream of the scpB gene and the insertion of IS1548 within the hylB gene. PFGE clustered the isolates into 3 main groups. One PFGE group accounted for 80% of typeable cerebrospinal fluid (CSF) isolates, versus 24% of colonization isolates (P=1.8 x 10-9). GBSi1 was found in 67% of CSF isolates and in only 23% of colonization isolates (P=5.3 x 10-7). A 15-kbp SmaI restriction-DNA fragment bearing the neuA gene was significantly associated with CSF isolates (P=1.1 x 10-11).