Background/Objectives: There are several biomarkers for measuring iron deficiency (ID) in pregnancy, but the prevalence of ID and its association with inflammation and adverse pregnancy outcomes is inconclusive. The aim of this work was to describe the prevalence and determinants of first trimester ID and associations with pregnancy and birth outcomes. Subjects/Methods: A record-linkage cohort study of archived serum samples of women attending first trimester screening and birth and hospital data to ascertain maternal characteristics and pregnancy outcomes. Sera were analysed for iron stores (ferritin; µg/l), lack of iron in the tissues (soluble transferrin receptor (sTfR); nmol/l) and inflammatory (C-reactive protein (CRP); mg/dl) biomarkers. Total body iron (TBI) was calculated from serum ferritin (SF) and sTfR concentrations. Multivariate logistic regression analysed risk factors and pregnancy outcomes associated with ID using the definitions: SF<12 µg/l, TfR≥ 21.0 nmol/l, and TBI<0 mg/kg. Results: Of the 4420 women, the prevalence of ID based on ferritin, sTfR and TBI was 19.6, 15.3 and 15.7%, respectively. Risk factors of ID varied depending on which iron parameter was used and included maternal age <25 years, multiparity, socioeconomic disadvantage, high maternal body weight and inflammation. ID, defined by SF and TBI but not TfR, was associated with reduced risk of gestational diabetes mellitus (GDM). ID defined using TBI only was associated with increased risk of large-for-gestation-age (LGA) infants. Conclusions: Nearly one in five Australian women begin pregnancy with ID. Further investigation of excess maternal weight and inflammation in the relationships between ID and GDM and LGA infants is needed.