Data on saturated fatty acids (SFAs) in relation to metabolic function and glucose homeostasis remain controversial. Such data are lacking among pregnant women. We prospectively investigated objectively measured individual and subclasses of plasma phospholipid SFAs throughout pregnancy in relation to cardiometabolic markers and gestational diabetes mellitus (GDM) risk. Within the National Institute of Child Health and Human Development Fetal Growth Studies-Singleton Cohort of 2802 singleton pregnancies, 107 GDM cases were ascertained via medical record review and matched to 214 non-GDM controls on age, race/ethnicity, and gestational week (GW) at blood collection. Individual plasma phospholipid SFA concentrations were repeatedly measured throughout pregnancy at GWs 10-14, 15-26, 23-31, and 33-39 and also grouped into subclasses of even- or odd-chain SFAs. From GW 10, even-chain SFA concentrations were significantly higher among women who later developed GDM, whereas odd-chain SFAs were significantly lower among GDM cases compared with controls. At GWs 10-14, the SFA palmitic acid (16:0) was positively associated with impaired insulin resistance and cardiometabolic markers and the risk of GDM [adjusted OR comparing the highest with the lowest quartile (aORQ4-Q1): 4.76; 95% CI: 1.72, 13.10; P-trend = 0.001]. In contrast, odd-chain SFAs were inversely related to the previously mentioned markers and GDM risk [aORQ4-Q1 for pentadecanoic acid (15:0): 0.32; 95% CI: 0.11, 0.92; P-trend = 0.025; for heptadecanoic acid (17:0): 0.20; 95% CI: 0.07, 0.58; P-trend = 0.003]. Women with high (median or greater) even-chain SFA concentrations and low (less than median) odd-chain SFAs had a 9.43-fold (95%: CI 3.26-, 27.30-fold) increased risk compared with women with low even-chain and high odd-chain SFA concentrations. Similar results were observed at GWs 15-26. The study provided one of the first lines of evidence suggesting that circulating concentrations of SFAs varying by SFA chain length, as early as GWs 10-14, were significantly and differentially associated with subsequent risk of GDM. Our findings highlight the importance of assessing objectively measured, individual, and subclasses of SFAs to investigate their distinct biological and pathophysiologic roles in glucose homeostasis and cardiometabolic outcomes. This study was registered at www.clinicaltrials.gov as NCT00912132.