AbstractDiabetes is a metabolic disorder of glucose homeostasis in which β cell destruction occurs silently and is detected mainly when symptoms appear. In the last few years, it has emerged a great interest in developing markers capable of detecting pancreatic β cell death focused on improving early diagnosis and getting a better treatment response, mainly in type 1 diabetes. But other types of diabetes would also benefit from early detection of β cell death. Differentially methylated circulating DNA is being studied as minimally invasive biomarker of cell death. We aimed to explore whether the unmethylated/methylated ratio of the insulin and amylin genes might be a good biomarker of β cell death in different types of diabetes. A lower index ∆Ct indicates a higher rate of β-cell death. Plasma samples from subjects without diabetes, pregnant women, pregnant with gestational diabetes (GDM), type 1 diabetes and type 2 diabetes were analyzed. A qPCR reaction with specific primers for both methylated and unmethylated fragments of insulin and amylin genes were carried out. Pregnant women, GDM and non- GDM, showed a higher β-cell death for both markers (∆INS = 3.8 ± 2.1 and ∆Amylin = 8.5 ± 3.6), whereas T1D presented lower rate (∆INS = 6.2 ± 2.1 and ∆Amylin = 10.7 ± 2.9) comparable to healthy subjects. The insulin methylation index was associated with the newborn birth weight (r = 0.46; p = 0.033) and with insulin resistance (r = -0.533; p = 0.027) in the GDM group. The higher rate of β-cell death was observed in pregnant women independently of their metabolic status. These indexes could be a good indicator of β cell death in processes caused by defects on insulin secretion, insulin action, or both.