Methylation is a biochemical process involving the addition of a methyl group (-CH) to various chemical compounds. It plays a crucial role in maintaining the homeostasis of the endothelium, which lines the interior surface of blood vessels, and has been linked, among other conditions, to coronary artery disease (CAD). Despite significant progress in CAD diagnosis and treatment, intensive research continues into genotypic and phenotypic CAD biomarkers. This review explores the significance of the methylation pathway and folate metabolism in CAD pathogenesis, with a focus on endothelial dysfunction resulting from deficiency in the active form of folate (5-MTHF). We discuss emerging areas of research into CAD biomarkers and factors influencing the methylation process. By highlighting genetically determined methylation disorders, particularly the polymorphism, we propose the potential use of the active form of folate (5-MTHF) as a novel CAD biomarker and personalized pharmaceutical for selected patient groups. Our aim is to improve the identification of individuals at high risk of CAD and enhance their prognosis.