Diabetes mellitus is one of the most common chronic metabolic disorders affecting people irrespective of age, gender and race. Many kinds of mutation in mitochondrial DNA have been reported to be associated to the development of diabetes mellitus (DM) along with other genetic factors and /or environmental factors. Among several mtDNA mutations, rRNA, tRNA, and ND1 genes are linked to high susceptibility to DM. This study is conducted to investigate the prevalence of these three genes mutation in selected south Indian population. This study suggests that G3316A and T3202C might contribute to the development of DM. The A3384G and C3254A are found to be non-pathogenic mutations in the studied population. With the admit of limited studies conducted on genetic mutations and their influences in Indian diabetic population, this investigation might help to determine genotype–phenotype correlation for DM in south Indian population and also help in determining the role of mitochondrial mutation in the genetic predisposition of T2DM in this population.