Stress is an important and modifiable determinant of health, and its association with hair cortisol concentrations (HCC) during pregnancy remains unclear. We selected a random sample of 97 participants from a cohort of pregnant participants attending prenatal clinics in Lima, Peru. Each provided a hair sample at enrollment (mean gestational age = 13.1 weeks) and again at full-term delivery. Hair samples were segmented to reflect HCC in preconception and each trimester. At enrollment, measures of stress included: difficulty accessing basic goods, educational attainment, exposure to violence, fair or poor general health, perceived stress, and symptoms of depression, general anxiety, and post-traumatic stress disorder. Linear mixed models evaluated the association between each stress measure and absolute and relative changes in HCC. Pearson correlation coefficients (r) assessed correlations between HCC and continuous stress scores. Educational attainment of ≤12 years was associated with higher HCC in preconception and the 1st trimester, and general anxiety with lower preconception HCC. When modeling HCC patterns across the 4 hair segments, an educational attainment of ≤12 years was associated with higher HCC, high perceived stress with lower HCC, and general anxiety with steeper increases in HCC (group by time p value =.02). Only preconception HCC and GAD scores correlated (r = −0.22, p =.04). We observed few associations between stress and HCC. However, those that were seen were generally restricted to the preconception and 1st trimester. Further investigations into the association between stress and changes in HCC across pregnancy are warranted, and should include the preconception where possible.