Human cytomegalovirus (HCMV) infections remain a neglected public health issue. The aim of the present study was to evaluate the frequency of HCMV congenital infections in newborns up to 1 month in the Sao Paulo State, from 2010 to 2018. The molecular characterization of HCMV-positive samples was also undertaken. Urine samples from 275 potential congenital HCMV-infected patients were tested by real-time Polymerase Chain Reaction (qPCR). HCMV-positive samples were amplified by conventional PCR targeting the UL89 gene, sequenced and searched for mutations. A total of 32 (11.6%) positive-HCMV cases were detected (mean Ct 30.59); mean and median age of 10.3 and 6 days old, respectively. Children aged between 0-3 weeks had higher HCMV detection rates (84.4%; 27/32). UL89 gene was successfully sequenced in two samples, both classified as the human betaherpesvirus 5. No described resistance-associated mutations were identified. A routine screening in newborns coupled with the genetic characterization of key viral genes is vital to decrease sequels associated with congenital HCMV infections.