ABSTRACTThe prevalence and severity of many diseases differs by sex, potentially due to sex-specific patterns in DNA methylation. Autosomal sex-specific differences in DNA methylation have been observed in cord blood and placental tissue but are not well studied in saliva or in diverse populations. We sought to characterize sex-specific DNA methylation on autosomal chromosomes in saliva samples from children in the Future of Families and Child Wellbeing Study, a multi-ethnic prospective birth cohort containing an oversampling of Black, Hispanic and low-income families. DNA methylation from saliva samples was analysed on 796 children (50.6% male) at both ages 9 and 15 with DNA methylation measured using the Illumina HumanMethylation 450k array. An epigenome-wide association analysis of the age 9 samples identified 8,430 sex-differentiated autosomal DNA methylation sites (P< 2.4 × 10−7), of which 76.2% had higher DNA methylation in female children. The strongest sex-difference was in the cg26921482 probe, in theAMDHD2gene, with 30.6% higher DNA methylation in female compared to male children (P< 1 × 10−300). Treating the age 15 samples as an internal replication set, we observed highly consistent results between the ages 9 and 15 measurements, indicating stable and replicable sex-differentiation. Further, we directly compared our results to previously published DNA methylation sex differences in both cord blood and saliva and again found strong consistency. Our findings support widespread and robust sex-differential DNA methylation across age, human tissues, and populations. These findings help inform our understanding of potential biological processes contributing to sex differences in human physiology and disease.