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Objectives: This study aimed to compare polymerase chain reaction (PCR) and IgM detection using enzyme linked immune-sorbent assay (ELISA) in diagnosis of congenital cytomegalovirus (CMV) infection., Methods: This study was conducted from May 2009 to December 2010. Urine and blood samples were collected from 94 neonates with suspected congenital CMV infection. Serum and part of urine samples were stored at -20°C freezer, until the serologic and PCR tests were achieved. A 94 fresh urine samples were processed for cell culture. Nineteen (20.2%) out of 94 urine samples were proven positive for CMV infection by viral culture. For comparing PCR and IgM ELISA we used tissue culture technique as a reference, the 19 positive samples on culture (CMV group) and 20 negative samples (control group) were included in the comparison. Some characteristics of CMV and control groups were compared including sex, age, birth weight, gestational age < 37 and small for gestational age. Clinical and laboratory abnormalities were also compared in both groups., Results: This study showed that the sensitivity and specificity of PCR in relation to viral culture were 100% and 100% respectively, there was excellent agreement between both tests (Kappa coefficient was 1 and P=0.000). On the other hand, the sensitivity of IgM CMV ELISA in relation to viral culture was 63.2% and the specificity was 85%. There was good agreement between both tests (Kappa coefficient was 0.48 and P=0.002). By comparing CMV and control groups, there were high statistically significant differences between both groups as regard the birth weight, gestational age < 37 and small for gestational age items (P= 0.00, 0.03 and 0.01 respectively). There were statistically insignificant differences as regarding the clinical and laboratory abnormalities detected for neonates of both groups. In this study jaundice (63%) and hepato-splenomegaly (42%) were the most common clinical signs in both groups., Conclusions: PCR is more sensitive and specific technique for detection of congenital CMV infection than CMV IgM ELISA. Being more cost effective, less cumbersome and less time consuming in relation to viral culture, PCR may be used in detection of congenital CMV infection.