Human African trypanosomiasis (HAT) has been targeted for zero transmission to humans by 2030. Animal reservoirs of gambiense-HAT could jeopardize these elimination goals. This study was undertaken to identify potential host reservoirs for Trypanosoma brucei gambiense by detecting its natural infections in domestic animals of Chadian HAT foci. Blood samples were collected from 267 goats, 181 sheep, 154 dogs, and 67 pigs. Rapid diagnostic test (RDT) and capillary tube centrifugation (CTC) were performed to search for trypanosomes. DNA was extracted from the buffy coat, and trypanosomes of the subgenus Trypanozoon as well as T. b. gambiense were identified by PCR. Of 669 blood samples, 19.4% were positive by RDT and 9.0% by CTC. PCR revealed 150 animals (22.4%) with trypanosomes belonging to Trypanozoon, including 18 (12%) T. b. gambiense. This trypanosome was found in all investigated animal species and all HAT foci. Between animal species or villages, no significant differences were observed in the number of animals harboring T. b. gambiense DNA. Pigs, dogs, sheep and goats appeared to be potential reservoir hosts for T. b. gambiense in Chad. The identification of T. b. gambiense in all animal species of all HAT foci suggests that these animals should be considered when designing new control strategies for sustainable elimination of HAT. Investigations aiming to decrypt their specific role in each epidemiological setting are important to achieve zero transmission of HAT.