Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with complex heritability and higher prevalence in males. The neonatal epigenome has the potential to reflect past interactions between genetic and environmental factors during early development and influence future health outcomes. Methods: We performed whole-genome bisulfite sequencing of 152 umbilical cord blood samples from the MARBLES and EARLI high-familial risk prospective cohorts to identify an epigenomic signature of ASD at birth. Samples were split into discovery and replication sets and stratified by sex, and their DNA methylation profiles were tested for differentially methylated regions (DMRs) between ASD and typically developing control cord blood samples. DMRs were mapped to genes and assessed for enrichment in gene function, tissue expression, chromosome location, and overlap with prior ASD studies. DMR coordinates were tested for enrichment in chromatin states and transcription factor binding motifs. Results were compared between discovery and replication sets and between males and females. Results: We identified DMRs stratified by sex that discriminated ASD from control cord blood samples in discovery and replication sets. At a region level, 7 DMRs in males and 31 DMRs in females replicated across two independent groups of subjects, while 537 DMR genes in males and 1762 DMR genes in females replicated by gene association. These DMR genes were significantly enriched for brain and embryonic expression, X chromosome location, and identification in prior epigenetic studies of ASD in post-mortem brain. In males and females, autosomal ASD DMRs were significantly enriched for promoter and bivalent chromatin states across most cell types, while sex differences were observed for X-linked ASD DMRs. Lastly, these DMRs identified in cord blood were significantly enriched for binding sites of methyl-sensitive transcription factors relevant to fetal brain development. Conclusions: At birth, prior to the diagnosis of ASD, a distinct DNA methylation signature was detected in cord blood over regulatory regions and genes relevant to early fetal neurodevelopment. Differential cord methylation in ASD supports the developmental and sex-biased etiology of ASD and provides novel insights for early diagnosis and therapy.