Classical Hodgkin lymphoma (HL) exhibits a bi-modal age distribution that suggests an infectious aetiology. However, most cases of nodular sclerosis HL (NSHL) are Epstein–Barr virus (EBV) negative (60–90%). Previous studies regarding human herpesvirus 6 (HHV-6) positivity of HL have led to conflicting results. In order to clarify this situation, we examined NSHL biopsies for the presence and distribution of HHV-6 by immunohistochemistry (IHC), polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). PCR identified HHV-6 DNA in 86% of NSHL cases. As HHV-6 DNA was also identified in most cases of reactive lymphoid hyperplasia, we sought to localize the virus to specific cells by IHC, which detected HHV-6 in Reed–Sternberg (RS) cells of nearly half (48%) of NSHL cases. Dual CD30/HHV-6 immunostaining confirmed HHV-6 immunoreactivity in CD30+ RS cells, and HHV-6 PCR positivity was confirmed in laser capture microdissection-isolated CD30+ RS cells. FISH demonstrated multiple copies of HHV-6 genome in scattered cells. In contrast, EBV+ RS cells were identified in only 24% of the cases. HHV-6+ cases trended toward a younger age than EBV+ cases. These results conclusively demonstrate that RS cells in many cases of NSHL are HHV-6 positive, and suggest that HHV-6 may play a role in NSHL pathogenesis, particularly in younger patients with EBV-negative disease.