Background: Congenital cytomegalovirus (CMV) infection is a leading cause of hearing loss and mental delay. Infants may be asymptomatic at birth and may pass their Universal hearing test. Early identification of CMV-infected infants will allow earlier evaluation and management that may prove to be effective in reducing the risk of morbidity. The prevalence of congenital CMV infection in United States varies geographically from 0.5-5% and is unknown for our region. Objectives: Study the local prevalence of congenital CMV infection in asymptomatic newborns at a tertiary hospital in northeast Florida. Methods: The study was conducted at the University of Florida in Jacksonville between June 2016 and July 2017. It was approved by the Institutional Review Board. Inclusion criteria: age < 30 days, birth weight > 1750 grams, gestational age > 32 weeks at birth, and signed informed consent from the guardians. We excluded infants with any signs of congenital CMV disease as manifested by one or more of the following: thrombocytopenia, petechiae, hepatomegaly, splenomegaly, intrauterine growth restriction, hepatitis, microcephaly, sensorineural defects, imminent demise, prior or current receipt of ganciclovir, valganciclovir, foscarnet, cidofovir, brincidofovir, maribavir, or letermovir, maternal receipt of CMV hyperimmune globulin during pregnancy, or a breastfeeding mother receiving one of the above mentioned antivirals. Enrolled newborns were tested for the presence of urine CMV by nucleic acid amplification test (NAAT). Urine was collected via urine bag and transferred to the laboratory for testing according to laboratory protocols and test manufacturer recommendations. We reviewed the electronic health records of the enrolled newborns for collection of data. Data was analyzed to describe the demographics and to calculate the prevalence of asymptomatic CMV infection among our study population. Results: 104 newborns enrolled. Urine was successfully collected from 100 of 104 recruited infants. 4 infants without a urine sample were excluded. One infant had a positive urine NAAT for CMV making the prevalence of congenital CMV infection among asymptomatic newborns in our geographic location 1%. The CMV infected newborn was delivered at 41 weeks of gestation via spontaneous vaginal delivery to a 16-year-old primigravida woman who was incarcerated at the time of delivery. Conclusions: CMV prevalence is low in our setting (urban, tertiary hospital)