Congenital cytomegalovirus (CMV) infection is the most common congenital viral infection worldwide and a leading cause of neurodevelopmental abnormalities, including sensorineural hearing loss, microcephaly, and developmental delay in neonates. Transmission occurs primarily through vertical maternal–fetal infection during early pregnancy. Although most infected neonates are asymptomatic at birth, a significant proportion develop long-term neurological sequelae. In low- and middle-income countries, including Nigeria, routine screening for congenital CMV is rarely performed, and molecular epidemiological data remain limited. This study aimed to determine the molecular prevalence and characterize congenital CMV infection among neonates attending tertiary health institutions in Jos, Nigeria. A cross-sectional molecular study was conducted among neonates aged ≤21 days recruited from three tertiary hospitals in Jos between January 2021 and December 2022. Buccal swab samples were collected and tested for CMV DNA using standardized in-house polymerase chain reaction (PCR). Positive samples were subjected to Sanger sequencing, and phylogenetic analysis was performed. Data were analyzed descriptively, and molecular prevalence was reported with exact 95% confidence intervals. Out of 180 neonates enrolled, one tested positive for CMV DNA, giving a molecular prevalence of 0.6% (95% CI: 0.02–3.1%). BLAST analysis revealed 98.9% nucleotide sequence similarity to Human herpesvirus 5 strain HAN22, and the sequence was assigned the GenBank accession number PV668598. Phylogenetic analysis showed clustering with previously reported African isolates. The CMV-positive neonate presented with microcephaly and small-for-gestational-age status. Although congenital CMV infection was rare in this cohort, molecular detection and genomic characterization provide valuable baseline data on CMV epidemiology in Nigeria and underscore the importance of continued surveillance and early diagnosis.