Streptococcus agalactiae is the leading cause of sepsis in neonates and contributes to morbidity and mortality. Transmission of this commensal urogenital and gastrointestinal organism can occur during vaginal delivery from a colonised mother. A universal screening program has been implemented in Western Australia and other parts of the world to test pregnant mothers for S. agalactiae at 36 weeks gestation to eradicate the organism in those colonised prior to delivery. Current gold-standard screening is culture-based, however, this is not as sensitive as molecular methods and does not define serotypes; potentially important in terms of virulence. The strains associated with infant disease typically include cps types Ia, Ib, III. We aimed to develop and optimise a multiplex PCR method to determine the presence of S. agalactiae in combination with identification of clinically relevant serotypes Ia, Ib, and III. Current PCR methods in this area involve multiple steps or only detect single serotypes. Primers were developed using the cps gene regions which are unique to each serotype and the dltS gene which is ubiquitous among S. agalactiae. The assay was validated with control strains, and then with 512 vaginal samples collected from pregnant women at three time points during pregnancy. Of these, 109 (21%) were positive for S. agalactiae. 13.8% of positive samples were further identified as cps Ia and 5.5% as cps III. No samples were positive for cps Ib. In conclusion, we have developed and optimised a specific and sensitive method to detect S. agalactiae presence and serotypes Ia, Ib and III in a single real-time multiplex PCR assay. This assay is of significant clinical relevance in that it is highly sensitive for diagnostic applications and also provides epidemiological data on cps type, information that may be important for vaccine development and other targeted non-antibiotic treatment therapies.