Trypanosoma cruzi infection leads to Chagas disease (CD), a neglected tropical infection of significant public health importance in South and Central America and other, non-endemic, countries. Pregnant women and their children are of particular importance to screen as T. cruzi can be transmitted vertically. The objective of this study was to screen for T. cruzi infection among pregnant women from endemic areas seen at the Hospital General de Mexico for prenatal care, so that they and their children may be quickly connected to CD treatment. Pregnant women were recruited through the hospital prenatal clinic and screened for T. cruzi infection using a series of serological and molecular tests. Of 150 screened patients, mean age 26.8 (SD 6.4), 30 (20.0%) were positive by at least one diagnostic test. Of these, only nine (6%) were positive as determined by PCR. Diagnosis of chronic CD is difficult in endemic places like Mexico due to the limitations of current commercially available diagnostic tests. Further evaluation of diagnostic performance of various assays could improve current CD diagnostic algorithms and proper care management in these regions. Genetic variability in the parasite may also play a role in the differing assay performances seen in this study, and this may be a valuable avenue of further research.