Abstract Oncocytomas are mostly benign tumors of epithelial origin, characterized by dense accumulation of defective mitochondria. To date, no nuclear gene has been found to be responsible for these tumors, but a correlation of the oncocytic phenotype with the occurrence of disruptive mitochondrial DNA (mtDNA) mutations, mostly affecting genes encoding for proteins of Complex I of the respiratory chain (NADH dehydrogenase), has been shown in sporadic oncocytomas of different organs, including renal oncocytomas (RO). Kidney cancer is made up of a number of tumors that present with a different histology and clinical course. They are caused by different genes, and therefore therapy needs to be adapted for each type of renal tumor. Imaging techniques are usually not sufficient to distinguish between benign and malignant forms of renal cell cancer (RCC). In addition, it is difficult to differentiate benign ROs from malignant forms of RCCs in samples from biopsies, because they share ultrastructural appearances, including abnormal mitochondria with altered cristae. Therefore, a reliable diagnostic tool would be invaluable to guide clinicians in the management of patients with RCC of unclear phenotype. In our current work we tested whether mtDNA mutations can be used as diagnostic tools to distinguish between benign ROs and more aggressive forms of RCC. We sequenced the entire mtDNA in 36 samples that included sporadic, bilateral and multifocal as well as familial ROs and 30 non-oncocytic RCCs of different histologies. Moreover, the mitochondrial copy number was determined in all samples by real-time PCR. Furthermore, we sequenced the mtDNA from biopsies and compared it to the mtDNA sequence of the resected tumor. We found a strong correlation between the presence of disruptive mtDNA mutations affecting complex I genes and the oncocytic phenotype in renal tumors. Additionally, oncocytomas showed significantly higher mtDNA copy number when compared to other RCCs. We also show that the mtDNA sequences from the biopsies correspond to the sequence of the tumor itself and are predictive of the phenotype. Hence, we propose that sequencing of the mitochondrial genome, supported by the analysis of the mtDNA copy number, may be used as a diagnostic tool to distinguish between benign and aggressive forms of RCCs to guide physicians in choosing the appropriate treatment for patients. Citation Format: Martin Lang, Cathy D. Vocke, Maria J. Merino, Laura S. Schmidt, Marston W. Linehan. Mitochondrial DNA mutations are diagnostic markers of renal oncocytomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-107. doi:10.1158/1538-7445.AM2013-LB-107