OBJECTIVE To identify fetal cord blood prognostic markers of symptomatic congenital human cytomegalovirus infection (HCMV). METHODS Retrospective observational study. METHODS Fetal medicine unit in Milan and Medical virology unit in Pavia, Italy. METHODS HCMV-infected and -uninfected fetuses of mothers with primary HCMV infection during the period 1995-2009. METHODS Overall, 94 blood samples from as many fetuses of 93 pregnant women experiencing primary HCMV infection were examined for multiple immunological, haematological and biochemical markers as well as virological markers. Congenital HCMV infection was diagnosed by detection of virus in amniotic fluid, and symptomatic/asymptomatic infections were determined by ultrasound scans, nuclear magnetic resonance imaging, histopathology or clinical examination at birth. Blood sample markers were retrospectively compared in symptomatic and asymptomatic fetuses with congenital infection. METHODS A statistical analysis was performed to determine the value of each parameter in predicting outcome. RESULTS Univariate analysis showed that most nonviral and viral markers were significantly different in symptomatic (n = 16) compared with asymptomatic (n = 31) fetuses. Receiver operator characteristics analysis indicated that, with reference to an established cutoff for each marker, the best nonviral factors for differentiation of symptomatic from asymptomatic congenital infection were β(2) -microglobulin and platelet count, and the best virological markers were immunoglobulin M antibody and DNAaemia. β(2) -Microglobulin alone or the combination of these four markers reached the optimal diagnostic efficacy. CONCLUSIONS The determination of multiple markers in fetal blood, following virus detection in amniotic fluid samples, is predictive of perinatal outcome in fetuses with HCMV infection.