Metabolic fingerprinting of amniotic fluid from streptozotocin-induced diabetic pregnant rats was performed using Fourier transform-ion cyclotron resonance mass spectrometry. Some of the fetuses from the diabetic pregnant rats exhibited ventricular septal defect. The positive ion profiles of amniotic fluids from diabetes were different to those of the control rats. The alteration of biochemical composition in the diabetic amniotic fluid suggests the presence of potential biomarkers to indicate progression of malformation under the diabetic pregnancy.