Background: The risk of preterm birth (PTB) increases when experiencing stress during pregnancy. Chronic stress has been associated with a dysregulation of the hypothalamic-pituitary-adrenal axis, for which hair cortisol concentration (HCC) is a promising biomarker. However, previous studies on the association between HCC and PTB yielded inconsistent results. This systematic review and meta-analysis synthesized previous studies on the association between maternal HCC before and during pregnancy and spontaneous PTB. Methods: Data was extracted from N = 11 studies with k = 19 effect sizes retrieved from PubMed, Embase, Web of Science, CINAHL and citation searching by hand in June 2023 and updated in October 2023. Standardized mean differences were calculated, and a random-effects three-level meta-analysis was conducted. Effect heterogeneity was assessed using Q and I². Results: HCC during pregnancy was higher among PTB than term groups, but effects were not statistically significant (z = 0.11, 95% CI: − 0.28, 0.51, p = .54) and total heterogeneity was high (Q₁₆ = 60.01, p < .001, I²_Total = 92.30%). After leaving out two possible outlier studies in sensitivity analyses, HCC was lower among preterm compared to term delivering groups, although not statistically significant (z = − 0.06, 95% CI: − 0.20, 0.08, p = .39) but with a substantially reduced total heterogeneity (Q₁₂ = 16.45, p = .17, I²_Total = 42.15%). No moderators affected the estimates significantly, but an effect of trimester and gestational age at delivery is likely. Conclusion: There is currently no evidence of prenatal HCC differences between PTB and term groups as effects were small, imprecise, and not significant. Low statistical power and methodological weaknesses of the small-scale studies challenge possible biological inferences from the small effects, but further research on HCC during pregnancy is highly encouraged.