Human cytomegalovirus (HCMV), a ubiquitous herpesvirus, causes a lifelong subclinical infection in healthy adults but leads to significant morbidity and mortality in neonates and immunocompromised individuals. A region (referred to as UL/b') present in the Toledo strain of HCMV and low passage clinical isolates contains 19 additional genes, which are absent in the highly passage laboratory strain AD169. One of these genes, UL149 open reading frame, was amplified by PCR and sequenced from isolates obtained from infants with congenital HCMV infection, to determine whether genetic variation of this gene could influence the signs of the virus infection. The major finding is that the UL149 is a variable gene in all 26 clinical isolates, and the sequences from clinical isolates were classified into three major groups. It is concluded that the HCMV UL149 sequence is variable at the nucleotide level and it might play an important role in HCMV infection.