Objective To quantify human cytomegalovirus (HCMV) DNA in the blood and urine of infants of different ages with suspected HCMV infection, and in the breast milk of their mothers, and to evaluate the significance of HCMV DNA detection in the three specimen types in the diagnosis of HCMV infection among infants of different ages. Methods A total of 170 infants with suspected HCMV infection were divided into groups A (<28 days; n=43) and B (28 days to 5 months; n=127) according to their ages. Blood and urine were collected from the infants, and breast milk was collected from their mothers. The specimens were examined by fluorescence quantitative PCR for detection of HCMV DNA. Results In group A, HCMV DNA detection rates in blood, urine and breast milk were 65.1%, 18.6% and 93.0% respectively. In group B, HCMV DNA detection rates in blood, urine and breast milk were 64.6%, 92.9% and 72.4% respectively. HCMV DNA detection rate in urine in group B was significantly higher than in group A (P<0.01), however, HCMV DNA detection rate in mothers' breast milk in group B was significantfly lower than in group A (P<0.01). Among the 82 infants with positive results for blood and urine, the copy number of HCMV DNA in urine was significantly higher than that in blood. Conclusions HCMV DNA detection rates in urine and breast milk are different among infants of different ages, so use of suitable specimens according to age is of great significance for improving detection rate.