Abstract Background Human cytomegalovirus (HCMV) is the most widespread cause of congenital infection. The effects of various viral strains and viral loads on the infection outcome have been under debate. Objectives To determine the distribution of gN variants in HCMV strains isolated from children with congenital or postnatal infection and to establish the relationship between the viral genotype, the viral load, and the sequelae. Study design The study population included congenitally HCMV-infected newborns and children with postnatal or unproven congenital HCMV infection. The genotyping was performed by RFLP analysis of PCR-amplified fragments, and the viral load was measured by quantitative real-time PCR. Results Our results demonstrated that the HCMV genotypes gN3b, gN4b, and gN4c were prevalent in the patients examined. There were no differences in the distributions of gN genotypes in the congenitally and postnatally infected children. Multiple HCMV strains were detected in both groups of children. A significant association between the HCMV gN4 genotype and the incidence of neurological disorders was observed ( p = 0.045). Our results suggest that the detection of the gN2 or the gN4 genotype may be indicative of serious manifestations in children. In contrast, the gN3b and the gN1 genotypes represent less pathogenic HCMV strains. The HCMV load in urine was significantly higher in children with congenital infection compared with children with postnatal infection. No correlation was found between the viral load and the genotype. Conclusion Our results suggest that the gN genotype may be a virological marker of symptomatic HCMV infection in newborns.