Glucose-6-phosphate dehydrogenase (G6PD) in humans is in X-linked disorder, housekeeping enzyme and vital for the survival of every cell. It catalyses the oxidation of glucose-6-phosphate to 6-phospho Gluconat in the first committed step of the pentose phosphate pathway, which provides cells with pentoses and reducing power in the form of NADPH. NADPH is required to protect the cells against oxidative damage. The aim of this study was the molecular analysis of common G6PD mutations in the provinces of the Fars and Isfahan. The Study of G6PD deficiency was performed on 96 patients with a history of favism, consisted of 34 samples from Fars and 62 samples from Isfahan. Genomic DNA with specific oligonucleotide primers followed by digestion with restriction enzymes for known mutations such as; Mediterranean, Chatham, Cosenza, Aures, A-(202G-A/376A-G were carried out. The most common allele in Iran was found to be the G6PD Mediterranean (82.3%), followed by the G6PD Chatham (8.3%) and none of the samples had Cosenza or A-(G202A/A376G) mutation, and remained unknown (9.4%). Further sequencing required to search for the other mutations among the remained samples (9.4%).