Human cytomegalovirus (CMV) is a major cause of morbidity in fetuses following intrauterine infection. The glycoprotein (g) envelope trimeric gH/gL/gO and pentameric gH/gL/pUL128/pUL130/pUL131A complexes are required for CMV entry into fibroblasts and endothelial/epithelial cells, respectively, and both are targets for neutralizing antibodies. The role of sequence variability among viral strains in the outcome of congenital CMV infection is controversial. Variation in the CMV UL75 gene encoding glycoprotein H (gH), the UL115 (gL), the UL74 (gO), and the UL128 locus (UL128L) encoding three structural proteins (pUL128, pUL130, and pUL131A) was determined in 82 newborns with congenital CMV infection and 113 infants with postnatal or unproven congenital CMV infection. Genotyping was performed by sequencing analysis of PCR-amplified fragments and the PCR-restriction fragment length polymorphism (RFLP) method, and the viral load was measured by quantitative real-time PCR. The obtained results demonstrated that (1) different CMV variants and mixed CMV infections can be detected in newborns infected congenitally; (2) the gH1 genotype, UL130 variant 6, and UL131A variant 1 were associated with some signs/symptoms within cohort of pediatric patients, mainly consisting of infants with symptomatic CMV infection. The results revealed that pUL130, pUL131A, and gH polymorphisms seemed to be associated with the outcome of CMV infection in infants.