NGS technology obviates the need for cloning procedures by the generation of enormous amounts of short sequence reads starting from minimal input material. The benefits of NGS for HCMV genomics were first demonstrated through the elucidation of variants present in laboratory preparations of the AD169 and Towne strains. In an attempt to evaluate the effectiveness of NGS with clinical HCMV isolates, Cunningham et al. compared a more traditional PCR-based amplification and Sanger sequencing approach with a NGS approach using the Illumina Genome Analyzer. In addition, the 454 GS FLX platform was successfully used to determine the first complete genome sequence of an Asian HCMV isolate. Cunningham et al. showed that sequencing of complete HCMV genomes directly from clinical material is achievable, but given the small fraction of viral DNA, not practically amenable to high-throughput. In order to achieve a high-throughput application with NGS technology, a protocol to amplify and isolate highly pure viral DNA is desirable. Currently, 33 complete HCMV sequences are available in the NCBI GenBank, including 17 derived from unpassaged or moderately passaged material. Additional sequences of clinical isolates are necessary to better apprehend the genetic diversity and coding capacity of HCMV strains. Since sequencing complete genomes of clinically representative HCMV isolates in high-throughput awaits new amplification protocols, we have developed a dedicated amplification, sequencing and analysis workflow for HCMV genome characterization. The workflow maximizes sequencing capacity through the generation of highly pure HCMV DNA. The efficiency of using 454 GS FLX and/or IGA for HCMV full genome sequencing was compared. Using a series of validation experiments, we show that consensus sequences derived by the workflow are representative for the strain present in the original clinical isolate. The presented workflow enables high-throughput analysis of HCMV full genome sequences and could serve as an important tool in elucidating the genetic diversity of this complex herpesvirus. Viral and cellular DNA contents were evaluated using a quantitative PCR assay. HCMV DNA was quantitated through amplification of a fragment of the conserved major capsid protein-encoding gene UL86. For human DNA, a region of the bglobin household gene was amplified. Primers and probes were obtained from Eurogentec ; the sequences are listed in Table S1. The qPCR was carried out using TaqMan Universal PCR Master Mix on an Applied Biosystems 7500 Fast Real-Time PCR system, following the manufacturer’s protocols. Both standards and samples were quantitated in duplicate, viral and cellular DNA was quantitated in separate wells. For absolute quantitation, standard series were produced by serial dilution of HCMV UL86 and human b-globin standards. The standards were prepared through PCR amplification of the qPCR targets and products were gel purified using the QIAquick Gel Extraction Kit.