Mitochondrial diseases can be caused by pathogenic variants in nuclear or mitochondrial DNA-encoded genes that often lead to multisystemic symptoms and can have any mode of inheri-tance. Using a single test, Genome Sequencing (GS) can effectively identify variants in both genomes, but it has not yet been universally used as a first-line approach to diagnosing mitochondrial diseases due to related costs and challenges in data analysis. In this article, we report three patients with mito-chondrial disease molecularly diagnosed through GS performed on DNA extracted from blood to demonstrate different diagnostic advantages of this technology, including the detection of a low-level heteroplasmic pathogenic variant, an intragenic nuclear DNA deletion, and a large mtDNA dele-tion. Current technical improvements and cost reductions are likely to lead to an expanded routine diagnostic usage of GS and of the complementary “Omic” technologies in mitochondrial diseases.