Cholestatic infants are associated with congenital abnormalities or viral infections, such as cytomegalovirus (CMV) infection. CMV can be detected by polymerase chain reaction (PCR) in body fluids, including urine which can be obtained easily and is non-invasive. The objective was to detect CMV in urine specimens of cholestasis infants and to analyze its correlation with serological status. This was a descriptive observational study with the cross-sectional approach, used urine from 39 cholestatic infants who meet the inclusion and exclusion criteria and have been approved by Ethics Committee. The nested-PCR was performed from extracted urine and unextracted direct urine. Serological data of immunoglobulin (Ig) M and IgG data were collected. Data were analyzed by Chi-square. Detection of CMV from extracted urine by PCR showed positive in 87.2% patients and from unextracted urine was positive in 48.7% patients. Serological status showed that IgM was positive in 41.0% patients and IgG was positive in 89.7% patients. The acute infection (IgM+ IgG+) was found in 41.0% patients, past infection (IgM-IgG+) was 48.7% patients, and not infected (IgM-IgG-) was in 10.3% patients. The acute infection (IgM+ IgG+), past infection (IgM-IgG+) and not infected (IgM-IgG-) was found in 41.0%, 48.7%, and 10.3% patients, respectively. The correlation between PCR CMV from extracted urine with serological CMV was moderate, while the unextracted urine was low. It indicates that to detect the infection of CMV, PCR technique is more accurate than serological testing, and the extracted urine is more appropriate specimen as PCR template than direct urine.