Objective To explore the relationship between human cytomegalovirus （HCMV） UL144 sequence variability and clinical disease. Methods HCMV UL144 open reading frame （ORF） was amplified by PCR assay in 72 lowpassage isolates [65 con-genitally infective children and 7 healthy children who were HCMV-DNA positive by quantitative PCR （qPCR）]. All positive PCR products were analyzed by heteroduplex mobility assay and single-stranded conformation polymorphism （HMA-SSCP） and 32 of them were sequenced. Results Fifty-five patient isolates and five healthy children isolates were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORF. Phylogenetic analysis indicated that the nucleotide sequences could be separated into 3 major genotypes. Comparing between UL144 se-quences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among microcephaly and jaundice isolates respectively. Conclusions HCMV-UL144 existed in most of low passage isolates and sequences were hypervariable. The UL144 ORF and its predicted product with the high level of sequence variability in different kinds of isolates suggest that UL144 ORF might play a role in HCMV infectivity and subsequent diseases.