Migraine is a complex neurological disorder that may have monogenic or polygenic aetiologies. Some families have strong aggregation of the disorder, which could be due to the presence of deleterious large effect genetic variants, and some families display maternal inheritance which may be evidence of mitochondrial inheritance. Migraine is highly prevalent in patients with primary mitochondrial disorders, suggesting that impaired energy production and increased oxidative stress in the brain may contribute to susceptibility. In this study we investigated variants in the mitochondrial genome in migraine families. Next generation sequencing of mitochondrial genomes was conducted in 120 individuals from 30 families that feature migraine with aura in the pedigrees. Variants are annotated using the MitoMaster software, filtered to retain those with a minor allele frequency of <0.001, and run through HmtDB which to determine likely pathogenicity. Twelve variants of interest were identified and tested for segregation in the families and whether they were present in an independent migraine case-control population. An Ala to Thr missense variant in MT-CO1, predicted to be likely pathogenic, was found to be significantly associated with migraine, with a frequency of 0.023 in cases vs 0.0063 in controls (n=1570; OR=3.81; CI 1.41 to 10.30; p=0.0039). MT-CO1 encodes the mitochondrial cytochrome C oxidase I, a key enzyme in the inner mitochondrial membrane that catalyses the final step of oxidative phosphorylation. Modelling using I-TASSER suggested the Ala to Thr variant affected the predicted protein structure. Our findings provide evidence that mitochondrial dysfunction may increase susceptibility to migraine, possibly via energy metabolism deficits or increased oxidative stress.