Background: Acute respiratory tract infections (ARTI) are a major threat in children all around the world. Adenovirus is one of the most important pathogens causing respiratory tract infections in children. Adenovirus (AdV) respiratory tract infections is caused mainly by species B, C and E. Accurate and rapid identification of AdV respiratory tract infection is important to avoid unnecessary antibiotic prescription and prevents AdV-related outbreaks. Objectives: This study aimed to identify the most prevalent species of adenovirus causing ARTI in pediatric patients hospitalized at Benha University Hospital which were diagnosed by tissue culture and direct immunofluorescence (DIF) test and compare them with polymerase chain reaction (PCR) as a gold standard method. Methodology: This study was conducted on 60 pediatric patients suffering from ARTI admitted to Benha University Hospitals. Adenovirus was detected by DIF test and isolated by tissue culture. Molecular identification of species- specific adenovirus was done by PCR. Results: Out of 60 cases, 35% were Adenovirus positive by PCR. AdV-B was the most prevalent identified species (76.2%) followed by AdV-C (19.0%) and AdV-E (4.8%). The sensitivity and the specificity of tissue culture and DIF test were 90.5%, 100%, 61.9%, 97.4%, respectively. Conclusion: AdV was encountered in one-third of admitted pediatric Egyptian patients with ARTI in Benha University Hospital. PCR was useful for rapid diagnosis of adenovirus infections with higher sensitivity than other methods.