Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, affects 8-10 million people worldwide and represents a major public health challenge. There is no effective treatment or vaccine to control the disease that is characterized by a mild acute phase followed by a chronic life-long infection. Approximately 30% of chronically infected individuals develop cardiac and/or digestive pathologies. T. cruzi can invade a wide variety of nucleated cells, but only persists at specific tissues in the host. However, the mechanisms that determine tissue tropism and the progression of the infection have not been fully described. Identification of infection niches in animal models has been difficult due to the limited quantity of parasite-infected cells and their focal distribution in tissues during the chronic phase. To better understand the course of chronic infections and parasite dissemination, we developed a bioluminescence imaging system based on the use of transgenic T. cruzi Colombiana strain parasites expressing nanoluciferase. Swiss Webster mice were infected with luminescent trypomastigotes and monitored for 126 days. Whole animal in vivo imaging showed parasites predominantly distributed in the abdominal cavity and surrounding areas throughout the infection. Bioluminescence signal reached a peak between 14 to 21 days post infection (dpi) and decreased progressively over time. Total animal luminescence could still be measured 126 dpi while parasites remained undetectable in blood by microscopy in most animals. Ex vivo imaging of specific tissues and organs dissected post-mortem at 126 dpi revealed a widespread parasite distribution in the skeletal muscle, heart, intestines and mesenteric fat. Parasites were also detected in lungs and liver. This noninvasive imaging model represents a novel tool to study host-parasite interactions and to identify parasite reservoirs of chronic Chagas Disease.