Research shows that serum Vitamin B12 levels are reduced in diabetes including gestational diabetes. We sought to compare serum cobalamin between pregnant females diagnosed with gestational diabetes mellitus (GDM) and healthy controls and to propose a cut off value for serum B12 levels as a predictive biomarker for GDM. Study design was Quantitative cross-sectional study. This study was carried out at Lady Atchison Hospital/Mayo Hospital Lahore, Pakistan from January to December 2016. Cobalamin levels were measured in women diagnosed with GDM (n=59) and controls (n=41). GDM was diagnosed when fasting blood sugar levels were 95 mg/dL or above. The levels were compared using Mann Whitney U test. Multiple linear regression was used to investigate the association of cobalamin with fasting blood sugar level after adjusting for gestational age and gravidity. ROC curve analysis was conducted to assess the suitability cobalamin as a predictive biomarker for GDM. Median cobalamin were significantly reduced in cases (108.95±92 pg/mL) as compared to controls (173±90 pg/mL) at p<0.001. Low vitamin B12 levels (β = -0.29, p=0.004) significantly predicted high fasting blood sugar levels in pregnancy even after adjusting for gestational age and gravidity. ROC curve analysis revealed the optimal cutoff point of serum cobalamin for predicting gestational diabetes to be 113 pg/mL at which sensitivity and specificity were was 56.9% (95%CI 44.1-68.8%) and 80.5 % (95%CI 65.6-89.9%) at P<0.0001. It was concluded that low serum cobalamin levels may be a risk factor and may predict the development of GDM.