NOTCH pathways are important regulators at the beginning of the genetic mechanisms controlling the embryonic development and cell differentiation required for normal pancreatic development during the embryonic period. Disruption of the NOTCH pathway induces apoptosis in pancreatic cells or corrupted pancreatic development that can lead to diabetes. Genetic mutations affecting the NOTCH pathway have been extensively studied in certain types of cancer, including solid tumours, but its metabolic functions are not well known. The objective of our study was to explore the relationship between NOTCH3 gene variants and the risk of developing type 2 diabetes mellitus in 100 patients and 100 healthy control subjects. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was carried out to identify the genotypes in 100 patients with diabetes and 100 control individuals. DNA extracted from peripheral blood samples was amplified and the rs1043994 variant was digested with MwoI, while the rs3815188 variant was digested with AciI. The products were evaluated by 3% agarose gel electrophoresis. The obtained results suggested that the rs1043994 variant was associated with the development of type 2 diabetes due to a significant ratio with the presence of the A allele. Similarly, the presence of the CC genotype and the absence of the T allele were determined to be associated with a notable risk of developing type 2 diabetes for individuals with the rs3815188 variant. In conclusion, we found a significant association between the rs1043994 and rs3815188 variants of the NOTCH3 gene and the risk of developing type 2 diabetes mellitus.