Background: Pyruvate kinase (PK) enzyme deficiency is an autosomal recessive disorder with both male and female equally affected. It is the most common enzyme abnormality in the glycolytic pathway of red blood cell metabolism. Clinical presentation of PK deficiency ranges from hydrops fetalis to mild compensated hemolytic jaundice. Objective: The aim of this work was to determine prevalence of RBCs pyruvate kinase enzyme deficiency among neonates with pathological indirect jaundice in Sharkia Governorate. Patients and Methods: This Cross-sectional study was done in Neonatal Intensive Care Unit, Pediatric Department at Zagazig University Hospitals during the period between June 2018 and December 2018. This study included 95 patients with pathological indirect hyperbilirubinemia. Results: There was statistically significant positive correlation between total bilirubin and all of reticulocytes' count, and platelet count. There was statistically significant negative correlation between total bilirubin and all of hemoglobin, red blood cells (RBCs), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC). There was non-significant correlation between it and other parameters. Pyruvate kinase deficiency was diagnosed in 4.2% of 95 patients with indirect hyperbilirubinemia. Glucose-6-phosphate dehydrogenase (G6PD) deficiency was diagnosed in 16.8%.  Conclusion: Red blood cells (RBCs) pyruvate kinase enzyme deficiency among neonates with pathological indirect jaundice is the second enzyme deficiency cause indirect jaundice after G6PD and is a risk factor related to pathological jaundice in neonates.