To investigate the variability of human cytomegalovirus (HCMV) UL140 open reading frame (ORF) in clinical strains, and to explore the relationship between the variability of UL140 ORF and different symptoms of HCMV infection. HCMV UL140 ORF was amplified by polymerase chain reaction and sequenced selectedly in 30 clinical strains. UL140 ORF of all clinical strains was amplified successfully. Compared with that of Toledo strain, the nucleotide and amino acid sequence identities among all strains were 96.5%–100.0% and 95.2%–100.0%, respectively. All of the nucleotide changes were substitutions. The post-translational modification sites were conserved. The result of phylogenetic tree showed that the strains did not cluster according to different clinical symptoms. HCMV UL140 ORF in clinical strains is highly conserved, which may play an important role in HCMV infection.