Objective Although coenzyme Q10 (CoQ10) deficiencies are heterogeneous it is often associated with mitochondrial respiratory chain disorders (RCD), specifically complex II+III (CII+II) deficiencies. The aim of this study was to investigate the association of defects in the biosynthesis of CoQ10 and associated RCD in South African patients. Methods A method to quantify CoQ10 in muscle was developed and standardized using isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS-MS). The CoQ10 quantification was then performed on 155 muscle specimens of patients who clinically qualified for respiratory chain enzymes analyses. Of these patients, 76 had confirmed RCD of which 29 patients had CII+III deficiencies. Ion Torrent next generation sequencing (NGS) was performed on 24 patients, which included nine patients with decreased and 15 with normal CoQ10 levels. Eighteen targeted genes involved in primary as well as secondary CoQ10 deficiencies were sequenced. Results CoQ10 was deficient in nine patients of which eight patients had CII+III deficiency. Race, age and gender did not have any influence on the CoQ10 levels. A total of 16 possible disease-causing variants were identified. Three novel compound heterozygous variants in three patients (two with deficient CoQ10 levels) were found in the ETFDH, COQ6 and COQ7 genes respectively. Protein analyses supported pathogenicity of the variants in the ETFDH and COQ6 genes. Conclusion Only RCD patients with confirmed CII+III enzyme deficiencies had statistically significant low muscle CoQ10 levels and at least two patients had novel compound heterozygous mutations in the ETFDH and COQ6 genes.