Heteroplasmy, the mixture of mitochondrial genomes (mtDNA), varies among individuals and cells. Heteroplasmy levels alter the penetrance of pathological mtDNA mutations, and the susceptibility to age-related diseases such as Parkinson's disease. Although mitochondrial dysfunction occurs in age-related type 2 diabetes mellitus (T2DM), the involvement of heteroplasmy in diabetes is unclear. We hypothesized that the heteroplasmic mutational (HM) pattern may change in T2DM. To test this, we used next-generation sequencing, i.e. massive parallel sequencing (MPS), along with PCR-cloning-Sanger sequencing to analyze HM in blood and skeletal muscle DNA samples from monozygotic (MZ) twins either concordant or discordant for T2DM. Great variability was identified in the repertoires and amounts of HMs among individuals, with a tendency towards more mutations in skeletal muscle than in blood. Whereas many HMs were unique, many were either shared among twin pairs or among tissues of the same individual, regardless of their prevalence. This suggested a heritable influence on even low abundance HMs. We found no clear differences between T2DM and controls. However, we found ~5-fold increase of HMs in non-coding sequences implying the influence of negative selection (P < 0.001). This negative selection was evident both in moderate to highly abundant heteroplasmy (>5% of the molecules per sample) and in low abundance heteroplasmy (<5% of the molecules). Although our study found no evidence supporting the involvement of HMs in the etiology of T2DM, the twin study found clear evidence of a heritable influence on the accumulation of HMs as well as the signatures of selection in heteroplasmic mutations.