Background: Hearing loss (HL) is a common congenital disease present at birth, happening in approximately one to three of every 1000 healthy newborns. Evoked otoacoustic emission (OAE) is extremely useful in infant hearing screening. Tympanometry is a test that measures the changes of the acoustic impedance (AI) of the tympanic ossicular system (TOS). Objective: This study aimed to evaluate the role of high-frequency tympanometry (HFT) in neonatal hearing screening (NHS), and to study the prevalence of HL in a sample of newborns. Patients and methods: This Cross-sectional study included 100 neonates who were tested in three stages, first stage was screening by transient evoked otoacouastic emissions (TEOAE) and HFT at primary health care centers, second and third stages was audiological diagnosis at the Audiology Unit of Mansoura New General Hospital. Results: Tympanometry (1000Hz) was with type A in 152 ears and type B in 48 ears. Ipsilateral acoustic reflexes were present in 146 ears and absent in 54 ears. In 2^nd step, tympanometry (1000Hz) was type A in 29 right ears and 29 in left ears and type B in 8 right ears and 8 left ears. In 3^rd step, tympanometry (1000Hz) with type A was in 22 ears and type B in 4 ears. Normal click auditory brainstem response (ABR) threshold was found in 17 ears and abnormal click ABR threshold was found in 9 ears. Conclusion: 1000 Hz tympanometry has to be a component of the NHS test battery. The HFT has to be considered for NHS programs to detect the middle ear pathological conditions in infants.