Mitochondrial disorders causing respiratory chain dysfunction comprise a group of genetically and clinically heterogeneous diseases. This heterogeneity reflects both the biochemical complexity of oxidative phosphorylation and the genetic contribution of both the nuclear and mitochondrial genomes to the respiratory chain. Current approaches to diagnose and classify mitochondrial disorders incorporate clinical, biochemical, and histological criteria, as well as DNA-based molecular diagnostic testing. While the identification of pathogenic mutations is generally accepted as definitive, the large number of candidate nuclear genes, the involvement of two genomes, and potential heteroplasmy of pathogenic mitochondrial DNA (mtDNA) frequently complicate successful molecular diagnostic confirmation. The strategy for pursuing a diagnosis derives from the integration of family history, clinical findings, biochemical evaluations, histopathological analyses, neuroradiological results, and the availability of different tissues for analyses. Screening for common point mutations and large deletions in mtDNA is usually the first step. Specific subsets of known nuclear disease genes can be screened by direct sequencing for cases of recognizable patterns of respiratory chain deficiencies or clinically identifiable syndromic presentations. Measurement of mtDNA content in affected tissues such as muscle and liver allows screening for mtDNA depletion syndromes. The growing list of known disease-causing genes and the promise of next generation sequencing technologies will undoubtedly improve diagnostic accuracy and genetic counseling for this challenging group of disorders.