Sequences of a 2.6- to 2.9-kb open reading frame in a putative immediate-early region of the human herpesvirus 6 (HHV6) genome were determined and compared in three reference and six local isolates of HHV6. The sequences segregated into two variant groups (A and B) having only 75% nucleotide homology and 62% peptide homology, in part because of deletions in the variant A strains. Among the variant B isolates, further sequence grouping was evident; two clinical isolates segregated with reference strain Z29 into a cluster that had 96.6% nucleotide homology and 92.6% peptide homology when compared to a second variant B cluster of four isolates. Within each cluster of variant B isolates, nucleotide homology was 99.4% or more. Two pairs of isolates had identical sequences. The marked divergence of variants A and B permitted the design of variant-specific oligonucleotide primers that could detect in a single polymerase chain reaction the presence of either or both variant A and B HHV6 DNA. Variation in this gene region of HHV6 is more extensive than in the envelope glycoprotein (gB and gH) coding regions and could be related to known biological differences between variant groups.