Leishmanial forms of Trypanosoma cruzi with multifocal pseudocysts were identified in a deceased adult female rhesus monkey (Macaca mulatta) that had been maintained in an outdoor primate colony in southern Texas. Subsequent clinical investigation of the remainder of the colony identified 20 additional monkeys seropositive for T cruzi. Concurrent epizootiologic surveys confirmed the presence of sylvatic reservoirs and triatomine vectors of T cruzi infection and identified a natural T cruzi transmission cycle. Veterinarians associated with the research colony were informed of the potential for further T cruzi transmission and appropriate insect vector and reservoir animal control programs were implemented..