It is unknown what strategy would be most effective in treating postnatal cytomegalovirus disease pCMV in newborns with very low birth weight (VLBW). Therefore, there have to be methods developed to make screening easier. The goal of this study was to ascertain if maternal milk and newborn saliva can be utilized to consistently identify maternal cytomegalovirus (CMV) serostatus and detect infant pCMV acquisition. The study included moms of very low birth weight (VLBW) infants born between 2017 and 2020 and was prospective in nature. CMV immunoglobulin G (IgG) was measured in breast milk using a CMV glycoprotein B binding enzyme-linked immunosorbent assay and compared to IgG levels in the mother’s serum. Infants born to moms with a positive or unknown CMV serostatus had their mothers collect saliva and urine samples every 2 weeks. The results of a quantitative real-time polymerase chain reaction (PCR) test for CMV DNA in saliva were compared to those of a qualitative PCR test for CMV DNA in urine from a clinical laboratory. About 10 (or 9%) of the 108 infants in the trial who did not have congenital CMV contracted the virus. Around 70 moms had both milk and blood CMV serology data available. When it comes to determining a person’s CMV serostatus, detecting maternal milk antibodies had a sensitivity of 97.2% (95% CI: 85.5-99.9%) and a specificity of 91.2% (95% CI: 76.3-98.1%). About 76 infants had paired serially collected saliva and urine samples (n = 203) to analyze. The sensitivity of saliva PCR for detecting pCMV acquisition was 30% (95% CI: 6.7% 65.2%), and the specificity was 92.70% (95% CI: 88.1-96.0%). Breast milk from a healthy mother is a good source for testing CMV immunity. There was insufficient sensitivity in serial infant saliva tests to detect pCMV acquisition in premature newborns.