Symptomatic disease due to human cytomegalovirus (HCMV) has been increasingly recognized in low birth weight (LBW) premature infants. Breast milk has been identified as a potential source for these infections. At approximately 60 days of life a LBW, premature infant was diagnosed with HCMV sepsis syndrome, prompting further investigation of the source for HCMV acquisition. The infant had received a combination of both fresh and frozen breast milk from his mother throughout his first 2 months of life. We utilized a real-time PCR assay for HCMV DNA to retrospectively examine viral genome copy number in serial samples of stored, frozen breast milk. This analysis indicated an increase in breast milk viral load over the first 45 days of life, heralding the HCMV sepsis syndrome that was observed clinically. Real-time PCR may be a useful tool in the evaluation of HCMV viral load in breast milk.