AbstractStudies were conducted to determine whether HHV‐8 hyperactivity could be the consequence of the propensity of the host to multiple HHV‐8 infection. The aim of the present work was to investigate HHV‐8 intrahost genetic variability. HHV‐8 subgenomic DNA was amplified by PCR from patients infected with HIV, health care workers (HCW) and bone marrow transplant recipients (BMT), and from oral lesional tissues of AIDS‐Kaposi's sarcoma (KS) patients. As controls, blood from HIV‐negative health care workers, and the cell lines BC‐1, BC‐2, and BCP‐1 were used. Clones derived from amplicons originating from DNA fragments in open reading frame (ORF) 26 and ORF K1 were isolated. For each ORF, intra‐specimen nucleotide sequence differences were determined. The extent of HHV‐8 variation in clones derived from blood of patients infected with HIV was significantly higher than in blood from health care workers or post‐bone marrow transplantation patients or in AIDS‐KS tissue. Among the clones derived from the latter three categories of specimens, sequence variations were not significant. It is concluded that HIV‐infected individuals can have multiple of HHV‐8, but AIDS‐KS lesions are associated with infection by a single HHV‐8 variant or a small group of related variants. J. Med. Virol. 85:636–645, 2013. © 2013 Wiley Periodicals, Inc.