Aims Alterations in organic acid biomarkers from fatty acid and carbohydrate metabolism have been documented in type 2 diabetes patients. However, their association with gestational diabetes mellitus (GDM) is largely unknown. Methods Participants were 25 GDM cases and 25 non-GDM controls. Biomarkers of fatty acid (adipate, suberate and ethylmalonate) and carbohydrate (pyruvate, L-lactate and β-hydroxybutyrate) metabolism were measured in maternal urine samples collected in early pregnancy (17 weeks) using liquid chromatography-mass spectrometry methods. Logistic regression were used to calculate odds ratios (OR) and 95% confidence intervals (CI). Results GDM cases and controls differed in median urinary concentrations of ethylmalonate (3.0 vs. 2.3 μg/mg creatinine), pyruvate (7.4 vs. 2.1 μg/mg creatinine), and adipate (4.6 vs. 7.3 μg/mg creatinine) (all p-values <0.05). Women in the highest tertile for ethylmalonate or pyruvate concentrations had 11.4-fold (95%CI 1.10-117.48) and 3.27-fold (95%CI 0.72-14.79) increased risk of GDM compared with women in the lowest tertile for ethylmalonate and pyruvate concentrations, respectively. Women in the highest tertile for adipate concentrations, compared with women in the lowest tertile, had an 86% reduction in GDM risk (95%CI 0.02-0.97). Conclusions These preliminary findings underscore the importance of altered fatty acid and carbohydrate metabolism in the pathogenesis of GDM.