Polymerase chain reaction (PCR) as a diagnostic tool for congenital Chagas disease is scarcely used. To assess the usefulness of PCR, a study in neonates was carried out to compare PCR with standard methods; that is bloodstream parasites by microhematocrit, and/or seroconversion by two conventional procedures. Seventeen neonates, born to mothers with positive Trypanosoma cruzi serology were analyzed by serologic, parasitological and molecular procedures, in blood samples taken at birth, and when aged 2-4 and 9 months. For a better comparison of sensitivity between both parasitological methods (microhematocrit and PCR), the number of samples of neonates studied at birth extended to 121. In children followed-up until 9 months of age, a 100% correlation between PCR and routine diagnosis was found, with PCR showing its highest sensitivity from Month 2 onwards. Comparative analysis between both parasitological methods, on samples taken at birth, showed a higher sensitivity of PCR respect the microhematocrit (P < 0.0008).