Background: Point mutations and structural variations (SVs) in mitochondrial DNA (mtDNA) contribute to many neurodegenerative diseases. Technical limitations and heteroplasmy, however, have impeded their identification, preventing these changes from being examined in neurons in healthy and disease states. Methods: We have developed a high-resolution technique—Mitochondrial DNA Structural Variation Sequencing (MitoSV-seq)—that identifies all types of mtDNA SVs and single-nucleotide variations (SNVs) in single neurons and novel variations that have been undetectable with conventional techniques. Findings: Using MitoSV-seq, we discovered SVs/SNVs in dopaminergic neurons in the Ifnar1^−/− murine model of Parkinson disease. Further, MitoSV-seq was found to have broad applicability, delivering high-quality, full-length mtDNA sequences in a species-independent manner from human PBMCs, haematological cancers, and tumour cell lines, regardless of heteroplasmy. We characterised several common SVs in haematological cancers (AML and MDS) that were linked to the same mtDNA region, MT-ND5, using only 10 cells, indicating the power of MitoSV-seq in determining single-cancer-cell ontologies. Notably, the MT-ND5 hotspot, shared between all examined cancers and Ifnar1^−/− dopaminergic neurons, suggests that its mutations have clinical value as disease biomarkers. Interpretation: MitoSV-seq identifies disease-relevant mtDNA mutations in single cells with high resolution, rendering it a potential drug screening platform in neurodegenerative diseases and cancers. Funding: The Lundbeck Foundation, Danish Council for Independent Research-Medicine, and European Union Horizon 2020 Research and Innovation Programme.