Purpose: Stress and elevated maternal glycemia have negative effects on pregnancy. We evaluated the association of hair cortisol concentrations (HCC), a marker of chronic stress, with insulin resistance and gestational diabetes (GDM). Methods: In total, 527 women from Lima, Peru, provided a hair sample in the second trimester of their pregnancy to measure HCC using liquid chromatography–tandem mass spectrometry. Each 6 cm of hair captured HCC in early (T1=1–12 weeks) and midpregnancy (T2 = 13–24 weeks). GDM diagnosis was conducted in midpregnancy. Multivariable regression models adjusted for putative risk factorsincluding maternal sociodemographic factors, diabetes history, and hair characteristics, were used to estimate the association of HCC with GDM and various glycemic traits. Results: GDM was diagnosed in 122 (23%) women. Mean HCC across pregnancy was T1 = 3.7 (±3.4) pg/mg and T2 = 4.8 (±3.4) pg/mg. HCC was associated with increased log-transformed units of fasting insulin (T1 = 0.15 [0.03, 0.27], T2 = 0.17 [0.04, 0.30]), homeostasis model assessment for insulin resistance (T1 = 0.14 [0.01, 0.26], T2 = 0.17 [0.03, 0.30]), and homeostasis model assessment for β-cell function (T1 = 0.20 [0.05, 0.34], T2 = 0.20 [0.04, 0.36]), but not with GDM (T1 = 0.95 [0.63, 1.40], T2 = 1.11 [0.74, 1.67]). Conclusions: Elevated maternal HCC was associated with abnormal insulin homeostasis in pregnancy. Dysregulation of the hypothalamic–pituitary–adrenal axis, as reflected by high HCC, may also contribute to insulin resistance syndrome in pregnancy.