OBJECTIVE To investigate the relationships between psychological and physiologic measures of stress, mood and gestational age at delivery and preterm birth (PTB). METHODS This prospective cohort study recruited healthy women in the early second trimester, 18–45 years of age. Validated psychological measures of perceived stress, depressive symptoms, and anxiety were completed at 16, 22, 28, 34 and 40 weeks of gestation. Cortisol concentration was measured in maternal hair at 16, 28, and 40 weeks of gestation to approximate 1st, 2nd, and 3rd trimester levels of physiologic stress. Statistical methods included: analyses of variance (ANOVA), t-tests, Chi-square, Pearson correlations, regression modeling and mediation analysis as appropriate. Hair cortisol concentrations were natural log transformed to normalize values. RESULTS Eleven (12%) out of 90 included women had a spontaneous preterm birth or preterm premature rupture of the membranes. Perceived stress at 16 weeks of gestation correlated with both 2nd trimester cortisol concentration (r= 0.28, p=0.007) and earlier gestational age at delivery (r= −0.30, p< 0.01). Gestational age at delivery was also negatively correlated with cortisol concentration in the 2nd trimester (r= −0.25, p=0.02) and 2nd trimester cortisol concentration was higher in preterm (2.7 ± 0.4 LN pg/mg) vs term (2.0 ± 0.7 LN pg/mg, P<0.001) delivered women. Using mediation statistics, the association between the psychological measure, the physiologic measure, and gestational age at delivery was mainly driven by increased physiologic stress (hair cortisol concentration) in the 2nd trimester (difference in coefficients (standard error)= −0.05(0.02)),. CONCLUSION Higher perceived stress in the 2nd trimester is associated with both elevated 2nd trimester hair cortisol concentration and gestational age at delivery. Physiologic measure of stress in the 2nd trimester appears most strongly associated with preterm birth. Identification and amelioration of early pregnancy stressors may attenuate physiologic stress and ultimately affect preterm birth.