Bats (Chiroptera) are the only mammals naturally able to fly. Due to this characteristic they play a relevant ecological role in the niches they inhabit. These mammals spread infectious diseases from enzootic to domestic foci. Rabbies, SARS, fungi, ebola and trypanosomes are the most common pathogens these animals may host. We conducted intensive sampling of bats from the phyllostomidae, vespertilionidae and emballonuridae families in six localities from Casanare department in eastern Colombia. Blood-EDTA samples were obtained and subsequently submitted to analyses of mitochondrial and nuclear genetic markers in order to conduct barcoding analyses to discriminate trypanosome species. The findings according to the congruence of the three molecular markers suggest the occurrence of Trypanosoma cruzi cruzi (51%), T. c. marinkellei (9%), T. dionisii (13%), T. rangeli (21%), T. evansi (4%) and T. theileri (2%) among 107 positive bat specimens. Regarding the T. cruzi DTUs, we observed the presence of TcI (60%), TcII (15%), TcIII (7%), TcIV (7%) and TcBAT (11%) being the first evidence to our concern of the foreseen genotype TcBAT in Colombia. These results allowed us to propose reliable hypotheses regarding the ecology and biology of the bats circulating in the area including the enigmatic question whether TcBAT should be considered a novel DTU. The epidemiological and evolutionary implications of these findings are herein discussed.