The majority of human herpesvirus 6 (HHV-6) congenital infections (86%) originate from germ line transmission of chromosomally integrated HHV-6 (ciHHV-6). To determine whether transplacentally acquired HHV-6 could derive from the transmission of reactivated maternal ciHHV-6, we identified mother-infant pairs in which infants had proven transplacentally acquired HHV-6 and mothers had documented ciHHV-6, and we sequenced and compared the HHV-6 gB gene sequences for each pair. Our data indicate that the gB gene sequence found in each cord blood specimen was identical to that of the corresponding mother but divergent from that of other known HHV-6 isolates. These results are consistent with transplacentally acquired HHV-6 originating from the transmission of reactivated ciHHV-6.