Chromosomal integration of human herpesvirus 6 (HHV-6) is a novel situation found in a small percentage of individuals. While active HHV-6 infection is treatable using antivirals, the abnormally high level of HHV-6 DNA found in chromosomal integration of HHV-6 (CI-HHV-6) is not affected by such drugs. Stored DNA samples taken originally for detection of fusion genes and minimal residual disease from 339 pediatric patients treated for leukemia in the Czech Republic between the years 1995-2007 were tested retrospectively. Using real-time quantitative PCR technology, the quantity of HHV-6 DNA detected was normalized to 100,000 human genome equivalents as assessed by quantitation of the albumin gene. HHV-6 DNA was detected in 107 samples from 91 patients (26.8%). In the majority of samples (99) only a minute level of normalized viral copies (NVCs) (median 1.84 NVCs) was detected. A high viral load of approximately 100,000 NVCs was detected in 5 patients (1.5%; median 140,150 NVCs), in all of whom were confirmed subsequently CI-HHV-6 by a detection of HHV-6 DNA in hair follicles or in the nails. In all but one patient with HHV-6 variant B, variant A of the virus was detected. None of the patients with CI-HHV-6 had complications attributable to HHV-6 infection. The prevalence of CI-HHV-6 in childhood leukemia does not differ from that published for other patients or healthy populations. Where high levels of HHV-6 DNA are present, CI-HHV-6 should be confirmed as soon as possible so that potentially toxic but ineffective antiviral treatment can be stopped.