--- - |2 Abstract The risk of developing posttraumatic stress disorder (PTSD) increases with the number of traumatic event types experienced (trauma load) in interaction with other psychobiological risk factors. The NOTCH (neurogenic locus notch homolog proteins) signaling pathway, consisting of four different trans‐membrane receptor proteins (NOTCH1–4), constitutes an evolutionarily well‐conserved intercellular communication pathway (involved, e.g., in cell–cell interaction, inflammatory signaling, and learning processes). Its association with fear memory consolidation makes it an interesting candidate for PTSD research. We tested for significant associations of common genetic variants of NOTCH1–4 (investigated by microarray) and genomic methylation of saliva‐derived DNA with lifetime PTSD risk in independent cohorts from Northern Uganda (N1 = 924) and Rwanda (N2 = 371), and investigated whether NOTCH‐related gene sets were enriched for associations with lifetime PTSD risk. We found associations of lifetime PTSD risk with single nucleotide polymorphism (SNP) rs2074621 (NOTCH3) (puncorrected = 0.04) in both cohorts, and with methylation of CpG site cg17519949 (NOTCH3) (puncorrected = 0.05) in Rwandans. Yet, none of the (epi‐)genetic associations survived multiple testing correction. Gene set enrichment analyses revealed enrichment for associations of two NOTCH pathways with lifetime PTSD risk in Ugandans: NOTCH binding (pcorrected = 0.003) and NOTCH receptor processing (pcorrected = 0.01). The environmental factor trauma load was significant in all analyses (all p < 0.001). Our integrated methodological approach suggests NOTCH as a possible mediator of PTSD risk after trauma. The results require replication, and the precise underlying pathophysiological mechanisms should be illuminated in future studies. - Psychophysiology, EarlyView.