The International Association of Diabetes and Pregnancy Study Groups recommend early testing for gestational diabetes (GDM) using fasting glucose (FG) of 5.1-6.9 mmol/L at the first prenatal visit. Nonetheless, the optimal approach to diagnosing GDM remains debatable given the limited data on glycemia in the first half of pregnancy. We evaluated the diagnostic performance of FG, insulin, and HOMA-IR in the first and second trimesters in prediction of GDM diagnosis in the third trimester. In a nested case-control study within the prospective Pregnancy Environment and Lifestyle Study (PETALS) pregnancy cohort, 193 GDM cases were diagnosed at gestational weeks 24-28 using the Carpenter-Coustan criteria and matched to 386 controls on age and race/ethnicity. FG, insulin and HOMA-IR were assessed at gestational weeks 10-13 and 16-19, respectively. The mean (SD) of FG and insulin were 4.4 (0.4) mmol/L and 8.4 (10.9) µU/mL at weeks 10-13, and 4.4 (0.5) mmol/L and 11.5 (18.3) µU/mL at weeks 16-19, respectively. The sensitivity of GDM diagnosis using FG of 5.1-6.9 mmol/L was 0.25 (95% CI 0.19-0.32) at gestational weeks 10-13, 0.29 (0.22-0.36) at weeks 16-19, and 0.39 (0.32-0.47) at either weeks 10-13 or 16-19; the corresponding specificity was 0.97 (0.94-0.98), 0.94 (0.91-0.96), and 0.91 (0.88-0.94). By lowering the cutoff to 4.9 mmol/L (90th percentile among controls) for FG, the corresponding sensitivity was 0.38 (0.31-0.45), 0.42 (0.35-0.49), and 0.54 (0.46-0.61) at gestational weeks 10-13, 16-19, or either, with specificity ranging from 0.81-0.90. Diagnostic performance of GDM was not enhanced by using fasting insulin or HOMA-IR. Our data show that only 25% or 29% of GDM can be diagnosed using FG ≥5.1 mmol/L at gestational weeks 10-13 or 16-19 and thus do not support a single FG value of 5.1-6.9 mmol/L in the first or second trimester as the criteria for GDM diagnosis. It is still imperative to conduct an OGTT at gestational weeks 24-28 to confirm or rule out GDM. Disclosure Y. Zhu: None. M.M. Hedderson: None. J. Feng: None. C. Quesenberry: None. A. Ferrara: None. Funding National Institutes of Health