Patients with Chagas cardiomyopathy carry a significant risk of reactivation after heart transplantation. Reactivation of Chagas disease can lead to graft failure or systemic complications such as fulminant central nervous system disease and sepsis. As such, careful screening for Chagas seropositivity prior to transplant is crucial to preventing negative outcomes in the post-transplant setting. One challenge in screening these patients is the variety of laboratory tests available and their differing sensitivities and specificities. In this case report, we present a patient who tested positive by a commercial Trypanosoma cruzi antibody assay and later tested negative by CDC confirmatory serological analysis. After the patient underwent orthotopic heart transplant, he underwent protocol-based polymerase chain reaction surveillance for reactivation as a result of persistent concerns for T. cruzi infection. It was discovered shortly thereafter that the patient had reactivation of Chagas disease, confirming that he did have Chagas cardiomyopathy prior to transplantation, despite negative confirmatory testing. This case illustrates the complexities of serological diagnosis of Chagas disease and the importance of additional testing for T. cruzi when the post-test probability remains high even with a commercial, negative serologic test.