Background: Due to the tropism of human parvovirus B19 to erythroid progenitor cells, infection in patients with an underlying hemolytic disorder such as thalassemia, hereditary spherocytosis, sickle cell disease and Glucose-6-phosphate dehydrogenase deficiency leads to suppression of erythrocyte formation, referred to as transient aplasia crisis (TAC), which may be life-threatening. Objectives: Detection of parvovirus B19 DNA and its IgG antibodies in the serum of children with chronic hemolytic anemia and in apparently healthy children in Benha University Hospitals. Methodology: The study was conducted on 80 children. Forty of them with chronic hemolytic anemia, they were subdivided into 2 groups, Group (1a) included 20 patients without history of aplastic crisis, Group (Ib) included 20 patients with a history of aplastic crisis and 40 age and sex-matched apparently healthy children representing control (Group II). All patients were subjected to full history taking, clinical examination and laboratory investigations. Parvovirus B19 IgG was measured using anti-parvovirus B19 ELISA kits (SUNRED), and parvovirus B19 DNA was detected by using nested-polymerase chain reaction. Results: The seroprevalence of parvovirus B19 IgG was significantly higher (P value =0.016) in Group Ia (50%) (10 out of 20) and Group Ib (45%) (9 out of 20) than the control group (Group II) (17.5%) (7 out of 40). There was a significant positive correlation between anti-parvovirus B19 IgG and age of all patients, frequency of blood transfusion. The prevalence of parvovirus B19 DNA was 10% (2 out of 20) in group Ia and 30% (6 out of 20) in group Ib and no viral DNA was detected in the controls (P value=0.001). Although 42.3% (11 out of 26) of children with β thalassemia major had a detectable level of antiparvovirus B19 virus IgG antibodies, only (23.1%) (6 out of 26) of them had B19 DNA. Anti-parvovirus B19 IgG antibodies were detected in 4 children out of 5 children of sickle cell anemia (80%) but the the prevalence of Parvovirus B19 DNA was 20% among them. Conclusion: Measures to keep away from iatrogenic and nosocomial infection transmission should be implemented including screening of donated blood for parvovirus B19 especially blood given to patients with blood disorders. Recommendation: Data from this study support the need for introduction of an approved vaccine that mainly protects children with chronic hemolytic anemia against that infection.