For effective control programs, accurate assessment of Trypanosoma cruzi infection in vectors is essential and has traditionally been performed by microscopic examination. For particular vectors and not others, polymerase chain reaction (PCR) analysis of fecal samples recently has been shown to be an effective means of detection. The sensitivities of the PCR and microscopy for detection of T. cruzi in different anatomic sites were compared in the two major vectors of Guatemala, Triatoma dimidiata and Rhodnius prolixus. Preliminary studies established that T. cruzi can be detected by the PCR in the presence of 90% T. rangeli. One hundred thirty-five vectors were collected, and samples were obtained from the rectum, intestines, and stomach and analyzed by microscopy and the PCR. For Triatoma dimidiata rectal samples, the PCR sensitivity (39.1% T. cruzi positive) and the microscopic sensitivity (24.6% positive) was not significantly different. However, in R. prolixus, the PCR proved significantly more sensitive than microscopy: 57.6% positive by PCR compared with 22.7% by microscopy. Rectal samples showed the highest rates of infection followed by intestine and stomach samples. However, 10.5% of the Rhodnius infections would have been missed if only the rectal sample had been analyzed. Thus, the PCR is significantly more sensitive than microscopy for detection of T. cruzi in R. prolixus. Analysis of anatomic sites in addition to the rectal sample may be necessary for accurate assessment of infection in particular vectors.