The genomes of commonly used variants of human cytomegalovirus (HCMV) strains Towne and AD169 each contain a substantial mutation in which a region (U(L)/b') at the right end of the long unique region has been replaced by an inverted duplication of a region from the left end of the genome. Using high-throughput technology, we have sequenced HCMV strain Towne (ATCC VR-977) and confirmed the presence of two variants, one exhibiting the replacement in U(L)/b' and the other intact in this region. Both variants are mutated in genes RL13, UL1, UL40, UL130, US1 and US9. We have also sequenced a novel AD169 variant (varUC) that is intact in U(L)/b' except for a small deletion that affects genes UL144, UL142, UL141 and UL140. Like other AD169 variants, varUC is mutated in genes RL5A, RL13, UL36 and UL131A. A subpopulation of varUC contains an additional deletion affecting genes IRS1, US1 and US2.