Mitochondrial disease refers to a heterogeneous group of disorders resulting in defective cellular energy production due to abnormal oxidative phosphorylation (oxphos). Primary mitochondrial disease (PMD) is diagnosed clinically and ideally, but not always, confirmed by a known or indisputably pathogenic mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) mutation. The PMD genes either encode oxphos proteins directly or they affect oxphos function by impacting production of the complex machinery needed to run the oxphos process. However, many disorders have the ‘mitochondrial’ phenotype without an identifiable mtDNA or nDNA mutation or they have a variant of unknown clinical significance. Secondary mitochondrial dysfunction (SMD) can be caused by genes encoding neither function nor production of the oxphos proteins and accompanies many hereditary non-mitochondrial diseases. SMD may also be due to nongenetic causes such as environmental factors. In our practice, we see many patients with clinical signs of mitochondrial dysfunction based on phenotype, biomarkers, imaging, muscle biopsy, or negative/equivocal mtDNA or nDNA test results. In these cases, it is often tempting to assign a patient’s phenotype to ‘mitochondrial disease’, but SMD is often challenging to distinguish from PMD. Fortunately, rapid advances in molecular testing, made possible by next generation sequencing, have been effective at least in some cases in establishing accurate diagnoses to distinguish between PMD and SMD. This is important, since their treatments and prognoses can be quite different. However, even in the absence of the ability to distinguish between PMD and SMD, treating SMD with standard treatments for PMD can be effective. We review the latest findings regarding mitochondrial disease/dysfunction and give representative examples in which differentiation between PMD and SMD has been crucial for diagnosis and treatment.