Coxsackievirus B3 can cause severe neonatal disease with high mortality. We present the first report of detection of coxsackievirus B3 in the mothers' milk of 2 severely infected neonates by culture and reverse transcriptase-polymerase chain reaction. Reverse transcriptase-polymerase chain reaction is a rapid and sensitive tool to detect coxsackievirus B3 in breast milk within few hours. By 12 to 14 days after onset of symptoms, coxsackievirus B3 could not be detected in the breast milk of the symptomatic mothers.