Mitochondrial DNA(mt DNA) variation has been implicated in many common complex diseases, but inconsistent and contradicting results are common. Here we introduce a novel mutational load hypothesis, which also considers the collective effect of mainly rare variants, utilising the Mut Pred Program.We apply this new methodology to investigate the possible role of mt DNA in two cardiovascular disease(CVD) phenotypes(hypertension and hyperglycaemia), within a two-population cohort(n = 363; mean age 45 ± 9 yrs). Very few studies have looked at African mt DNA variation in the context of complex disease, and none using complete sequence data in a well-phenotyped cohort. As such, our study will also extend our knowledge of African mt DNA variation, with complete sequences of Southern Africans being especially under-represented. The cohort showed prevalence rates for hypertension(58.6%) and prediabetes(44.8%). We could not identify a statistically significant role for mt DNA variation in association with hypertension or hyperglycaemia in our cohort. However, we are of the opinion that the method described will find wide application in the field, being especially useful for cohorts from multiple locations or with a variety of mt DNA lineages, where the traditional haplogroup association method has been particularly likely to generate spurious results in the context of association with common complex disease. Mitochondrial DNA (mt DNA) variation has been implicated in many common complex diseases, but inconsistent and contradicting results are common. Here we introduce a novel mutational load hypothesis, which also considers the collective effect of mainly rare variants, utilising the Mut Pred Program. We apply this new methodology to investigate the possible role of mt DNA in two cardiovascular disease (CVD) phenotypes (hypertension and hyperglycaemia), within a two-population cohort (n = 363; mean age 45 ± 9 yrs). Very few studies have looked at African mt DNA variation in the context of complex disease, and none using complete sequence data in a well-phenotyped cohort. As such, our study will also extend our knowledge of African mt DNA variation, with complete sequences of Southern Africans are especially The cohort rated low prevalence rates for hypertension (58.6%) and prediabetes (44.8%). We could not identify a significant function for mt DNA variation in associ ation with hypertension or hyperglycaemia in our cohort. However, we are of the opinion that the method said will find wide application in the field, is especially useful for cohorts from multiple locations or with a variety of mt DNA lineages, where the traditional haplogroup association method has been particularly likely to generate spurious results in the context of association with common complex disease.