Background: There are no detailed reports in the literature on maternal cytomegalovirus antibody screening for universal newborn hearing screening (UNHS) referral patients. We examined maternal cytomegalovirus antibody screening results and estimated the incidence of maternal primary cytomegalovirus infection among UNHS referral patients. Methods: During September 2013–March 2021, fresh urine samples were collected in the first week after birth from 98 neonates with UNHS referral results at 15 obstetrical institutions in Mie, Japan (the first hearing screening). We performed a real-time polymerase chain reaction analysis to detect cytomegalovirus DNA in the samples. Infants with \ge200 copies/mL of cytomegalovirus DNA were diagnosed with congenital cytomegalovirus (cCMV) infection. A second hearing screening was performed, and patients with positive results were sent to the otorhinolaryngologists for further examinations of congenital hearing loss. We calculated incidence rates (%) with 95% confidence intervals (CIs) for cCMV infection among patients with UNHS referral results and maternal primary cytomegalovirus infection among patients who underwent maternal cytomegalovirus antibody screening. Results: Among the 98 neonates with UNHS referral results (the first hearing screening), 5 were diagnosed with cCMV infection (incidence rate: 5.1%; 95% CI: 0.8–9.5). All five patients with cCMV had positive second hearing screening results and were sent to their otorhinolaryngologists. All five were diagnosed with congenital hearing loss, and four were diagnosed with congenital hearing loss secondary to cCMV infection. The remaining patient with cCMV infection was diagnosed with congenital hearing loss unrelated to cCMV infection. Of the 98 patients, 60 underwent maternal cytomegalovirus antibody screening. Among the 60 patients, six had maternal primary cytomegalovirus infection during pregnancy (incidence rate: 10.0%; 95% CI: 2.4–17.6). Of the six patients, four were positive for cytomegalovirus immunoglobulin (CMV Ig) G and IgM antibodies in maternal blood with low CMV IgG antibody avidity results during early pregnancy, while the remaining two had maternal CMV IgG antibody seroconversion during pregnancy. Conclusions: This is the first study to examine the maternal primary cytomegalovirus infection incidence rate in patients with UNHS referral results (the first hearing screening). We identified a 10-fold higher risk in this population (10.0%) than in the general population (0.98%).