The insulin () gene is the one of the most important genes involved in the pathogenesis of Type 1 Diabetes (T1D) after the Major Histocompatibility Complex genes. Studies addressing the issue of hyper- or hypo-methylation status of the gene promoter have reported inconsistent results. The majority of studies showed hypomethylation; however a few studies have shown hypermethylation at specific cytosine-guanosine (CpG) sites in the promoter region of the gene. The aim of the present study was to analyze the methylation status of the promoter region of the gene in Greek children and adolescents with T1D. A total of 20 T1D participants (mean diabetes duration of 6.15±4.12 years) and 20 age- and sex-matched controls were enrolled in the present study. DNA was isolated from whole blood samples, modified using sodium bisulfite and analyzed using PCR and electrophoresis. DNA was then pooled with highly reactive supermagnetic beads at similar molar quantities, submitted for library construction and finally sequenced using next-generation sequencing. The methylation profile at 10 CpG sites around the transcription start site (TSS) of the promoter was analysed and expressed as the mean ± standard deviation. The overall mean methylation in patients with T1D did not differ compared with the healthy controls. There was a statistically significant difference between the two groups in hypermethylation at position -345 (P=0.02), while a trend (P=0.06) at position -102 was observed. According to the results of the present study, increased methylation in the gene promoter at specific CpG sites around the TSS were already present in childhood T1D. These data may possibly serve as a guide towards the identification of a methylation pattern for detection of development of T1D in genetically predisposed children.