Background: Human cytomegalovirus (HCMV) is the most prevalent cause of congenital infections, non-hereditary sensorineural hearing loss and other serious neurological sequelae in childhood. Predicting the consequences of an in utero infection is hindered by the lack of a deep understanding of the pathogenesis of congenital HCMV infections. Viral and host factors may influence the outcome, and viral factors may include variability in genes encoding of the essential envelope glycoproteins (gB, gM, gN, gH, gL, gO and gUL128-gUL131A). Objectives: The aims of the current study were to (1) develop a deep-sequencing-based method to genotype HCMV glycoproteins in single and mixed genotype infections and (2) assess the genetic polymorphism of these glycoproteins in children with confirmed congenital and unconfirmed congenital or postnatal infections, immunocompromised patients and immunocompetent individuals to investigate whether specific genomic variants may be correlated with different forms of HCMV disease. Methodology: A multiplex polymerase chain reaction (PCR) coupled with MinION nanopore sequencing and a novel bioinformatics pipeline was used. Results: The ability of the protocol to detect single and mixed genotypes was evaluated using five HCMV laboratory strains and mock mixed samples that were prepared from these strains. This method accurately detected all the expected single genotypes in the five laboratory strains and low-frequency minor genotypes down to the abundance of 1-10% in the mock mixed infection samples with a sequence accuracy of > 99%. The genomic variants of the glycoprotein genes were investigated in 109 clinical isolates from the 3 clinical groups. To verify the sequencing results, a subset of the samples was also subjected to the restriction fragment length polymorphism and direct sequencing methods. The developed protocol detected HCMV with a limit of detection of 102-103 IU/ml with an accuracy of > 99%. The sequencing protocol detected some or all of the glycoprotein genes in 109 samples and differentiated the glycoprotein gene sequences at the subtype level. gB.1, gM.2, gN.4 (4d), gH.2, gL.4 and gO.1 (1a) were the most dominant single genotypes in the group of children with congenital/postnatal infections, and a significant correlation was only found for gM.2 and gH.2. While gB.1, gM.3, gN.4, gH.1 and gH.2, gL.4 and gO.2 were prevalent genotypes in the immunocompromised group, gB.1, gM.1, gN.1 and gN.4, gH.1, gL.4 and gL.3 and gO.1 were found in the immunocompetent group. Significant associations were found only for gM.3 and gO.2 in the immunocompromised group and only for gO.1 in the immunocompetent group. The sequences of the UL128, UL130 and UL131A genes demonstrated low levels of amino acid variability among a subset of 39 clinical isolates (0.6-3%, 0.5-2.4% and 0.8-1.6%, respectively). Variabilities were phylogenetically grouped. High rates of mixed genotypes were detected in all studied groups. Conclusion and future directions: Using a novel multiplex PCR and deep sequencing, it was found that high rates of mixed HCMV infections frequently occurred in children with congenital/postnatal infections and the other studied groups. The HCMV strains causing such infections were extremely diverse. The study results suggest that the incidence of mixed infections has been underestimated in previous studies. Understanding the role of mixed genotypes in influencing replication within the host could aid the development of a virologic prognostic marker of the consequences of congenital infections.