Introduction and Aim: Diabetes mellitus is a complex disease that is a major global health problem. It is defined as a chronic metabolic disease characterized by high blood glucose levels. Diabetes risk has been linked to genetic variations within mitochondrial DNA. The current study sought to identify and compare genetic variation (mutations) in ATPase genes in diabetic patients and healthy individuals.   Materials and Methods: This study included 100 individuals (50 T2DM patients and 50 healthy volunteers).  Peripheral blood samples were collected from each individual and DNA extracted. The mitochondrial mtATPase genes were amplified using specific primers by polymerase chain reaction. The amplified products were sequenced, and the sequences obtained analyzed for nucleotide changes.   Results: The ATPase gene sequence analysis revealed nine nucleotide changes, three of which (A9039G, G9092A, and G9100T) are classified as polymorphisms in the human mitochondrial genome database. Furthermore, we presented the first description of mutations in ATPase genes such as (A8586G, C8637T, G8813A, A8953G, C8981T and T9024G). The A9039G mutation changed the amino acid isoleucine to glycine, the G9100T mutation changed the amino acid serine to isoleucine, and the C8981G mutation changed the amino acid threonine to isoleucine. Most patients were found to predict haplogroup H2a (H2a2), in addition to having H1c (H1c3) and L1c(L1c2). The presence of genetic variations in mtATPase genes may be an inheritable risk factor for type 2 diabetes mellitus pathogenesis.   Conclusion: Variants in ATPase mitochondrial genes may be one of the risk factors associated with type 2 diabetes. Hence, documenting these mutations is clinically important in the possibility of diagnosing diabetes, as well as the likelihood that these mutations could be pathogenic.