Streptococcus agalactiae, or Group B Streptococcus (GBS), is a leading neonatal pathogen that causes sepsis, meningitis and pneumonia. Globally, strategies have been implemented to address vertical transmission, and in Western Australia (WA) culture-based screening at 35-37 weeks gestation is part of routine care to guide antibiotic administration. In this study we aimed to describe antenatal GBS colonisation within a large population in WA; this is a population that has received little attention in previous research. A cohort of 814 pregnant women attending antenatal clinics (2015-2017) provided self-collected vaginal and rectal swabs at ≤22 weeks (n = 814) and ≥33 weeks (n = 567) gestation. These were assessed for GBS presence using culture and PCR and serotyping was conducted using molecular methods. Lifestyle questionnaires and medical data were collected. We observed an overall GBS colonisation rate of 24%, with 10.6% of positive participants transiently colonised. Ethnicity (Aboriginal, Torres Strait Islander and African), maternal age ≥25 years, vitamin use, frequent sexual intercourse (≥5 times/week) and use of sex toys were associated with GBS colonisation following logistic regression analysis. The dominant serotypes identified were Ia (27.9%), III (20.9%), II (16.3%), V (15.8%), Ib (8.4%), VI (5.1%), IV (2.8%), NT (1.9), VIII (0.5%) and IX (0.5%) at visit one, with V (18.9%) preceding serotype II (18.2%) at visit two. Serotype VII was not detected. This is the first cohort study to assess GBS colonisation in WA pregnant women and will be highly beneficial for guiding future clinical practice and therapeutic options, in particular, selection of suitable vaccine candidates.