The blood serum lipid profile of women with Gestational Diabetes Mellitus (GDM) is still under study. There are no data on the serum lipid profile of GDM patients with more severe (insulin treated) compared to milder forms (diet treated) GDM. The aim of our study was to analyze the blood serum lipid profile of patients with milder versus more severe forms of GDM and to compare these findings with those of healthy pregnant women. This cross-sectional analytical study included 30 insulin-treated GDM, 30 diet-only GDM and 30 healthy pregnant women. Serum lipid was extracted from the 90 participants and their lipid profiles were analyzed by lipid fingerprinting using liquid-chromatography-mass spectrometry. A total of 143 parent ions were differentially represented in each of the three groups, belonging to the following classes: Glycerophospholipids, Sterol Lipids, Sphingolipids, Prenol Lipids, Fatty Acyls and Glycerolipids. There were significant differences in the lipid profiles of healthy pregnant women compared to GDM patients and also between milder versus more severe forms of GDM. There are marked differences in lipid fingerprinting between healthy pregnant women compared to those with GDM in the third trimester. Moreover, the lipid profile of women with more severe forms of GDM differs considerably from that of women with milder forms of GDM. These findings may be useful to help clarify the pathogenesis of milder and more severe forms of GDM.