Chagas disease, also known as American trypanosomiasis, is caused by the flagellate protozoan Trypanosoma cruzi. It is a significant health concern in South and Central America, where millions of people are infected or at risk of infection, and is an emerging health concern in the US. The occurrence of Chagas disease in natural environments is supported by mammal host species, but those primary species may vary based on geographic location. In South Texas, the primary host species for the disease is poorly understood, and required a field study to determine the spatial distribution of T. cruzi prevalence in free-ranging mammals. Our study objectives were to determine the spatial distribution and prevalence of T. cruzi parasites in free-ranging mammals. We compared T. cruzi prevalence among species, among vegetative communities, and among different topographies (i.e., floodplain versus upland). From December 2011 through December 2013, 450 blood and tissue samples from geolocated free-ranging wildlife mammal species were analyzed with the use of polymerase chain reaction to detect protozoan T. cruzi DNA. We also calculated mammal abundance with the use of mark–recapture methodology and recorded capture-site characteristics such as vegetation structure. We found that animals in grasslands had a significantly lower infection rate when summed across all species compared with animals in dense hardwoods and semi-improved woodlands (P=0.001). A higher percentage of infections were found in the lower-elevation floodplain—65% (28/43) of animals sampled, compared to upland areas—25% (9/36) of animals sampled. Our study suggested that common free-ranging meso-mammals supported T. cruzi in natural environments and are of public health concern in South Texas. Mitigation strategies should consider a range of management activities to include vegetation management, selective application of insecticides, and changes in human behavior in high-risk areas.