American trypanosomiasis and leishmaniases are diseases caused by protozoans of the Trypanosomatidae family. In Venezuela, although several endemic foci of both diseases coincide, there are no reports of coinfection in mammals. The molecular diagnosis of the coinfection T. cruzi-Leishmania spp. was done in 527 blood samples collected on filter paper of several species of mammals (Canis familiaris, Equus asinus, Didelphis marsupialis, Equus mulus, Rattus rattus, Equus caballus, Artibeus fraterculus, Felis catus, Sus scrofa, Bos taurus, Capra hircus and Sciurus granatensis) from the states Cojedes, Aragua, Anzoátegui, Guárico, Miranda and Capital District. The T. cruzi infection was determined through PCR amplification of DNA of kinetoplast minicircles (kDNA) and satellite DNA (sDNA). The Leishmania spp. infection was detected by Leishmania nested PCR (Ln-PCR), and ribosomal DNA internal transcribed spacer 1 PCR (ITS1-PCR). The percentage of infection by T. cruzi was 23.5%, by Leishmania spp. 12.9% and coinfection was 5.7%. D. marsupialis was the species with the highest percentage of infection for each parasitosis (T. cruzi 34.3%, Leishmania spp. 20.0%) and coinfection (14.3%). Anzoátegui was the state with the highest percentage of infection for each parasitosis (T. cruzi 64.9%, Leishmania spp. 64.9%) and coinfection (43.2%). Infections were determined in species not reported as natural reservoirs of T. cruzi (E. asinus and E. mulus) and of Leishmania spp. (E. mulus and S. scrofa). Coinfection was a frequent phenomenon in mammals in several co-endemic zones evaluated.