BACKGROUND: Maternal prenatal stress is associated with physiologic and adverse mental health outcomes in the offspring, but the underlying biologic mechanisms are unknown. We examined the associations of maternal perceived stress, including preconception exposure, with DNA methylation (DNAm) alterations in the cord blood buffy coats of 358 singleton infants. METHODS: Maternal perceived stress was measured prior to and throughout pregnancy in a cohort of women enrolled in Effects of Aspirin in Gestation and Reproduction Trial (EAGeR) trial. Perceived stress assessments based on a standardized Likert-scale were obtained in periconception (~2 months preconception and 2-8 weeks of gestation) and pregnancy (8-36 weeks of gestation). Cumulative perceived stress was estimated by calculating the predicted area under the curve of stress reported prior to and during pregnancy. DNAm was measured by the Infinium MethylationEPIC BeadChip. Multivariable robust linear regression was used to assess associations of perceived stress with individual CpG probes. RESULTS: Based on a 0 to 3 scale, average reported preconception and early pregnancy stress were 0.76 (0.60) and 0.67 (0.50), respectively. Average mid- to late-pregnancy stress, based on a 0 to 10 scale, was 4.9 (1.6). Neither periconception nor pregnancy perceived stress were associated with individual CpG sites in neonatal cord blood (all false discovery rate [FDR] >5%). CONCLUSION: No effects of maternal perceived stress exposure on array-wide cord blood neonatal methylation differences were found.