Human cytomegalovirus (HCMV) is the most frequent cause of congenital infection. Despite the fact that 5-17% of asymptomatic infected babies will develop late sequelae and, therefore, should be closely followed, most of these children will remain undetected, as screening of all newborns by viral culture is too expensive and no valid alternative has been widely accepted. The aim of this work was to demonstrate that pool testing can be used to screen HCMV congenital infection in newborns. For this purpose, a real-time PCR technique was tested in urine pools. This pool method was applied to all urine specimens from children received in the virology laboratory of the Centro Hospitalar Cova da Beira for diagnosis of HCMV infection for a period of 14 months. Ten out of the 160 urine samples were tested positive by shell-vial culture and were also detected by this pool method. Additionally, 100 urine specimens, collected in 2004 and culture negative for HCMV were included to test the specificity of this methodology, all of which were negative. In conclusion, these results suggest that urine pools can be used to detect HCMV-positive urines in children, with similar sensitivity and specificity when compared with the standard method. Because of the very significant reduction both in terms of labour and cost of testing materials, this methodology may represent a valid option for screening the HCMV congenital infection in newborns.