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Introduction Among metabolic disorders, gestational diabetes mellitus (GDM) is specified as hyperglycemia caused by glucose or carbohydrate intolerance defects. GDM is distinguished by oxidative stress, and has been connected to mitochondrial dysfunction. Previous studies have documented the relation between A12026G, A8344G and A3243G mutations in ND4, tRNALeu(UUR), and tRNALys genes in different modes of diabetes. Aim The purpose of this study was to investigate into the relationship between GDM women and common mitochondrial mutations including A12026, A8344G, and A3243G in Saudi women. Methods In this case-control study, we have opted 96 GDM and 102 non-GDM pregnant women and DNA was extracted using EDTA blood and based on specific primers, Polymerase Chain Reaction was followed and then Restriction Fragment Length Polymorphism (RFLP) analysis was performed. Restriction enzymes was cross-checked with Lambda DNA and 10% of the purified PCR products were performed the Sanger sequencing analysis to reconfirm the RFLP analysis of the studied results. Results None of the heterozygous and homozygous mutations were not observed in our study. All the subjects were turned to be homozygous normal genotypes. Conclusion This study confirms that A12026, A8344G, and A3243G mutations have no role in the Saudi women with GDM.