This investigation applied polymerase chain reaction (PCR) using 3 sets of Trypanosoma cruzi–specific primers to amplify DNA from 31 archived formalin-fixed and fresh-frozen raccoon hearts. PCR successfully amplified T. cruzi–specific sequences, with at least 1 primer set, from multiple sites within the myocardium of formalin-fixed and fresh-frozen raccoon hearts that had previously tested positive using enzyme-linked immunosorbent assay and indirect immunofluorescent antibody titer in the absence of positive hemoculture results. Trypanosoma cruzi DNA was most frequently amplified from the interventricular septum, right ventricle, and left atrium. In addition, T. cruzi DNA was amplified with all 3 primers in at least 1 raccoon that was hemoculture positive and 2 animals that were borderline negative for the T. cruzi antibody and hemoculture negative. The amplification of T. cruzi–specific DNA sequences in the presence of an elevated antibody titer and negative culture results suggests good sensitivity of this method for detecting the presence of the parasite in archival tissues.