The prevalence of a congenital bicuspid aortic valve (BAV) is 0.5 to 1% of the population. The condition arises from the fusion of two aortic valve leaflets and is commonly associated with progressive dilation of the aorta (aortopathy) which can lead to sudden and life-threatening complications including dissection and rupture. Diagnosis of asymptomatic BAV-associated aortopathy is challenging and, once identified, criteria for surgical intervention are poorly defined. A non-invasive assay to easily detect aortopathy and identify those patients at high risk of complications would be a significant advance. The methylation pattern of DNA is unique to each cell type because it regulates gene expression and determines cell specificity. Given their differing embryological origins, we hypothesized that unique methylation patterns exist for the aortic root, tubular ascending aorta, and proximal aortic arch. Identification of these differentially methylated regions (DMRs) in cell-free DNA (cfDNA) will provide a biomarker of aortopathy. We also hypothesized that DNA methylation in the aorta is affected by valve morphology and is related to the pathogenesis of aortopathy. To address our hypotheses, we completed three specific aims: Aim 1: Identification of region-specific DMRs from healthy human aorta tissue. Aim 2: In vitro validation of tissue specificity of aorta-specific DMRs. Aim 3: Identification of DMRs in aortic tissue from patients with aortopathy. We have created the first DNA methylation atlas of the human aorta, creating genome-wide methylation profiles of the aortic root, tubular ascending aorta and proximal aortic arch. We have shown that the DNA methylation pattern of the aortic root is distinct from the two regions of the ascending aorta. Unfortunately, we were not able to validate region-specific aorta DMRs for use in a cfDNA-based assay, and this likely reflects the inadequate density and breadth of probes in the Illumina EPIC array that we selected for this project. Finally, we identified different methylation patterns in regions of the aorta from patients with aortopathy and a BAV. In conclusion, DNA methylation appears to be important in defining the regions of the aorta, and this information can be further developed to identify novel biomarkers of aortic injury. DNA methylation also appears to be largely differentially affected in aortopathy, but further work is needed to determine if this is a primary or secondary effect.