Introduction: Transfer RNA (tRNA) genes in the mitochondrial DNA genome are crucial for protein synthesis. Dysfunctional mitochondria, due to tRNA mutations, impair insulin secretion as they fail to operate optimally. These tRNA mutations may also contribute to insulin resistance. Moreover, the loss of tRNA modifications can lead to pancreatic β cell dysfunction. The limited understanding of the prevalence of the A3243G mitochondrial gene mutation in familial type 2 diabetes mellitus (T2DM) spurred this investigation. Our objective was to explore the prevalence and clinical characteristics of the mitochondrial tRNA A3243G mutation in inherited T2DM. Methods:The study enrolled a total of 100 subjects, divided into two groups: 50 with paternal inheritance and 50 with maternal inheritance. Participants were recruited from the Department of Endocrinology and Metabolism, SS Hospital, IMS, BHU, and all were undergoing treatment for diabetes with a familial history of the disease. Comprehensive clinical, molecular, and biochemical evaluations were conducted on all patients. Mitochondrial DNA (mtDNA) genes were amplified using polymerase chain reaction (PCR) and subsequently sequenced as part of the molecular assessments. Results:Participants in the maternal inheritance (MDI) group had a mean age of 43.24 ± 5.83 years, slightly higher than 41.99 ± 6.39 years in the paternal inheritance (PDI) group. Baseline and biochemical parameters (Hb, FPG, and HbA1c) were similar between both groups. The A3243G mutation was found in 6% of MDI participants and 2% of PDI participants, indicating a higher prevalence in the maternal inheritance group. Most participants in both groups did not exhibit this mutation. Comorbidities such as polyneuropathy, visual impairment, and renal impairment were noted, although many reported no additional symptoms. Treatment strategies ranged from lifestyle modification alone to combinations of oral hypoglycemic agents, insulin, and lifestyle adjustments as necessary. Conclusion:The mitochondrial A3243G mutation was identified in 4% of the diabetic patients. The occurrence of the A3243G mutation in the mitochondrial tRNALeu(UUR) gene, while low, is consistent with findings from previous studies. However, expanding the study to include a larger cohort and screening for additional mtDNA mutations beyond A3243G could provide deeper insights into the epidemiological dynamics and prevalence of MIDD or mitochondrial diabetes mellitus (mtDM).