Mitochondrial diseases are highly heterogeneous on the clinical, biochemical, and genetic level. In the traditional diagnostic approach (“biopsy first”) the evaluation of the affected individual and his body fluids, combined with the analysis of the respiratory chain enzymes in muscle based the subsequent Sanger sequencing of single candidate genes (“from function to gene”). Within the past few years, next-generation sequencing techniques of leucocyte-derived DNA (e.g., exome sequencing), with a diagnostic yield of more than 40%, have become the first line routine technology. This implicates that the invasive muscle biopsy is performed less often, especially in children. Furthermore, in this “genetics-first” approach the detection of new candidate genes precedes functional evaluations (“from gene to function”) leading to reverse phenotyping of affected individuals. Here, we line out the value of muscle and other tissue biopsies in this “genetics-first” era. We describe when and why it is still needed. We create awareness of pitfalls in the genetic diagnostics of mitochondrial diseases still necessitating tissue biopsies. Finally, we discuss why tissue biopsies are required for confirmatory diagnostics, or for getting a biochemical diagnosis in patients with hidden variants not detectable by standard genetics.