In the field of human cytomegalovirus pathogenesis there is growing interest in analyzing recent clinical isolates rather than cell culture adapted laboratory strains. However, true low passage isolates are strictly cell associated prior to cell culture adaptation and only a minor fraction of cells are infected at low passage number. Both conditions hinder the preparation of pure viral DNA. To date, genetic analyses had been carried out mostly with supernatant associated cytomegalovirus. A rapid and simple method is described for preparation of viral DNA from low passage cell associated isolates with little cytopathogenic effect. The protocol is based on a combination of Triton X-100 lysis, nuclease treatment, and subsequent phenol chloroform extraction. Cellular background was reduced significantly to enable clear detection of all viral DNA fragments in restriction fragment length analysis. The method yielded DNA which was suitable for downstream applications like cloning of viral DNA fragments or transfection of genomic viral DNA. This method may facilitate genomic analyses of pathogenic cell associated recent cytomegalovirus isolates.