Objective To estimate the prevalence of cytomegalovirus (CMV) infections in newborn-mother pairs, neonates with sepsis, and infants with hydrocephalus in Uganda. Design and Methods Three populations: (1) newborn-mother pairs, (2) neonates with sepsis, and (3) infants (≤ 3 months) with non-postinfectious (NPIH) or postinfectious (PIH) hydrocephalus, were evaluated for CMV infection at three medical centers in Uganda. Quantitative PCR (qPCR) was used to characterize the prevalence of CMV. Results The overall CMV prevalence in 2498 samples in duplicate across all groups was 9%. In newborn-mother pairs, there was a 3% prevalence of cord blood CMV positivity and 33% prevalence of maternal vaginal shedding. In neonates with clinical sepsis there was a 2% CMV prevalence. Maternal HIV seropositivity (aOR, 25.20; 95% CI, 4.43-134.26; p= 0.0001), residence in Eastern Uganda (aOR, 11.06; 95% CI, 2.30-76.18; p=0.003), maternal age < 25 years (aOR, 4.54; 95% CI, 1.40-19.29; p=0.02), and increasing neonatal age (aOR, 1.08 for each day older; 95% CI, 1.00-1.16; p= 0.05), were associated risk factors for CMV in neonates with clinical sepsis. We found a two-fold higher maternal vaginal shedding in Eastern (45%) vs Western (22%) Uganda during parturition (n=22/49 vs. 11/50, Fisher's exact test, p=0.02). In infants with PIH, the prevalence in blood was 24% and in infants with NPIH it was 20%. CMV was present in the CSF of 13% of infants with PIH compared to 0.5% of infants with NPIH (n=26/205 vs. 1/194, p<0.0001). Conclusion Our findings highlight that congenital and postnatal CMV prevalence is substantial in this African setting and the long-term consequences are uncharacterized.