This study aims to identify novel markers for gestational diabetes (GDM) in the biochemical profile of maternal urine using NMR metabolomics. It also catalogs the general effects of pregnancy and delivery on the urine profile. Urine samples were collected at three time points (visit V1: gestational week 8-20; V2: week 28±2; V3 10-16 weeks post partum) from participants in the STORK Groruddalen program, a prospective, multiethnic cohort study of 823 healthy, pregnant women in Oslo, Norway, and analyzed using (1)H-NMR spectroscopy. Metabolites were identified and quantified where possible. PCA, PLS-DA and univariate statistics were applied and found substantial differences between the time points, dominated by a steady increase of urinary lactose concentrations, and an increase during pregnancy and subsequent dramatic reduction of several unidentified NMR signals between 0.5 and 1.1 ppm. Multivariate methods could not reliably identify GDM cases based on the WHO or graded criteria based on IADPSG definitions, indicating that the pattern of urinary metabolites above micromolar concentrations is not influenced strongly and consistently enough by the disease. However, univariate analysis suggests elevated mean citrate concentrations with increasing hyperglycemia. Multivariate classification with respect to ethnic background produced weak but statistically significant models. These results suggest that although NMR-based metabolomics can monitor changes in the urinary excretion profile of pregnant women, it may not be a prudent choice for the study of GDM.