The aim of this study was to compare an in-house real-time PCR assay, with bacterial culture as the reference, for the diagnosis of late onset group B Streptococcal (GBS) disease. This was a retrospective review. All children aged 7-90 days presenting to four paediatric centres that had a blood or CSF sample tested by GBS PCR were included. Of 7,686 blood and 2,495 cerebrospinal fluid (CSF) samples from patients of all ages received for PCR testing, 893 and 859 samples were eligible for the study, respectively. When compared to culture, the sensitivity of blood PCR was 65% (13/20) in comparison to the CSF PCR test which was 100% (5/5). Ten of 23 PCR-positive blood samples and 17 of 22 PCR-positive CSF samples were culture negative. The median threshold Ct values for culture-positive/PCR-positive CSF samples was lower than that of culture-negative/PCR-positive CSF samples (p = 0.08). Clinical details of 17 available cases that were culture negative/PCR positive were reviewed; seven were deemed to be definite cases, eight were probable and two were possible. The results showed that detection of GBS by PCR is useful for CSF samples from infants aged 7-90 days with suspected meningitis; however, analysis of blood samples by PCR is of limited value as a routine screening test for late onset GBS sepsis and should not replace bacterial culture.