The impact of Chagas disease is no longer restricted to endemic areas. The aim of this study is to evaluate a 2-year period of a vertical transmission screening program of Trypanosoma cruzi infection in a tertiary care hospital in Barcelona (Spain). Two enzyme-linked immunosorbent assays (recombinant and crude antigen) were performed in parallel to pregnant women at risk of T. cruzi infection. Discordant results were confirmed by a third diagnostic test. In the case of a positive result, the newborn was tested at birth and after 8 months of life. A total of 1,473 women met the inclusion criteria for the screening program with a resulting seroprevalence for T. cruzi of 3.5% (2.2-5.2% 95% confidence interval [95% CI]). One case of congenital infection was identified. Screening programs for vertically transmitted T. cruzi acute infection are beneficial in non-endemic areas for early detection and treatment of acute infection.