Abstract The replication of human herpesvirus-6 (HHV-6) DNA is catalyzed by the viral DNA polymerase pU38 and the processivity factor pU27 which stabilizes the enzyme on the DNA template. The genetic polymorphism of pU27 among 46 clinical strains of HHV-6 variant A or B and four strains resistant to antivirals was investigated. Overall, 28 amino acid changes (7.6%) and a two-amino acid deletion were identified among the 368 residues of pU27, when using the U1102 (variant A) sequence as the reference. Eleven amino acid changes (3.0%) specifically differentiated both variants. The median intravariant amino acid variability was 1.2% and 0.3% for A and B, respectively. Except for a single change, the pU27 sequence of multi-drug resistant HHV-6 strains was also conserved. Structural models of pU27 for variants A and B were derived from that of the human cytomegalovirus homologue pUL44, and showed either identical or very similar residues in the regions interacting with viral DNA polymerase and viral DNA molecule. As pU27 is both highly conserved and essential for viral replication, it might constitute an interesting target for antiviral chemotherapy.