Background: Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency variants is essential, especially since the biochemical characterization has lost its significance due to the individual variability. As a result, cases can be misdiagnosed. The present study was designed to determine the incidence of G6PD Mediterranean (Med) mutation among Egyptian children with G6PD deficiency as well as its molecular association with the G6PD 1311T silent polymorphism. Methods: Fifty full term male, Egyptian neonates, presented with neonatal hyperbilirubinemia were subjected to quantitative G6PD enzyme assay. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used for detection of G6PD Mediterranean mutation and G6PD 1311 silent polymorphism. Results: Qualitative assay of G6PD level revealed presence of 21 cases (42%) out of 50 cases with neonatal hyperbilirubinemia had G6PD deficiency. Within the G6PD deficient group, there were 10 cases (47.62%) had mild G6PD deficiency, 4 cases (19.05%) had moderate G6PD deficiency, and 7 cases (33.33%) had severe G6PD deficiency. In this study, all cases with gene mutation were G6PD deficient, where 33.33% (7/21) cases had G6PD Mediterranean mutation (563 C-T), and 28.57% (6/21) cases had G6PD Silent mutation at Position (1311 C-T). Conclusion: G6PD deficiency seems to be a relatively common cause of neonatal jaundice in Egyptian infants. Detection of this enzymopathy by measuring G6PD level and molecular analysis of gene mutations can give a clue for early diagnosis of G6PD deficiency, monitoring for possible jaundice, and consequent prevention of possible complication as kernicterus in neonatal period, and hemolytic crisis later on.