Recent biosurveillance findings at Joint Base San Antonio (JBSA), a large military installation located in south-central Texas, indicate the potential for vector-borne human Chagas disease. A cross-sectional study was conducted to determine the prevalence and seroprevalence of Trypanosoma cruzi infection in highest risk subpopulations on the installation, including students and instructors who work and sleep in triatomine-endemic field settings. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and indirect immunofluorescent antibody assay were performed on enrolled subjects (N = 1,033), none of whom tested positive for T. cruzi or anti-T. cruzi antibodies. Current countermeasures used during field training on JBSA appear to be sufficient for preventing autochthonous human Chagas disease.