Background : Mitochondrial dysfunction is one of the most common causes of pediatric Encephalomyopathies. Mitochondrial (mt) and Nuclear (n) Deoxyribonucleic acid (DNA) mutations are important causes of encephalomyopathies. Objectives : This study aimed at assessment of the frequency of these mutations among 15 Egyptian children clinically diagnosed as mitochondrial Encephalomyopathies. Also, to highlight the various molecular techniques recruited to reach the molecular diagnosis in these disorders. Subjects and Methods : Open muscle biopsy was conducted in 9 patients. Total blood DNA was extracted from the patients and their families. Muscle DNA extraction was done in 2 patients. Histochemical staining for Complex IV and II was done in the muscle biopsies in 2 patients. Biochemical analysis of the activities of the respiratory chain enzymes was determined in the muscle biopsy homogenate in 4 patients. Molecular testing including Polymerase chain reaction/Restriction Fragment Length Polymorphism (PCR/RFLP), Southern Blot and Sequence analysis of the coding genes of the nDNA and mtDNA was conducted in 9 patients. Results : Histochemical analysis results revealed negative staining for Complex IV and positive staining for Complex II in 2 patients. Biochemical analysis results revealed combined Complex I and IV deficiency in 2 cases and Isolated complex I deficiency in 1 patient. The mt/n DNA ratio was 40% reduced in one patient confirming the diagnosis of Mitochondrial Depletion Syndrome (MDS). Direct sequencing of the candidate genes didn’t reveal any pathogenic mutations. Conclusions : The molecular testing for the most common point mutations as a preliminary diagnostic guidance in well defined mitochondrial syndromes is the least invasive and least expensive way. Also, targeted molecular diagnosis based on biochemical analysis of respiratory chain enzymes make the molecular evaluation of pediatric mitochondrial disorders easier. Negative results of molecular diagnosis for mtDNA genes doesnot exclude mitochondrial disorder and involvement of nuclear genes should be always considered.