Isolated complex I deficiency (OMIM: 252010) is a common biochemical phenotype observed in pediatric mitochondrial disease. The associated clinical and genetic heterogeneity is vast—individuals can present with a plethora of clinical symptoms ranging from isolated myopathy to Leigh syndrome (OMIM: 256000). Complex I is the first and largest complex of the mitochondrial respiratory chain and comprises 45 structural subunits with a minimal contingent of 20 ancillary proteins required for assembly and/or biogenesis.1 The genes encoding these proteins involve either the mitochondria’s own genetic material (mtDNA) or a nuclear-encoded gene of mitochondrial function,2 and to date, causative defects have been identified in 39 genes encoding either complex I structural subunits or assembly factors.3, 4 Additionally, complex I deficiency can occur as a secondary consequence of dysfunction involving alternative mitochondrial processes.5, 6 Application of next-generation sequencing in the form of panel-based target capture or whole-exome sequencing has already demonstrated its utility in the diagnosis of heterogeneous conditions such as mitochondrial disease and has been the catalyst for disease-associated gene discovery through its application to undiagnosed cohorts.7 Facilitated by the GeneMatcher tool,8 we report the findings from four unrelated, clinically affected children who presented with symptoms suggestive of mitochondrial disease.