Crithidia mellificae, a monoxenous trypanosomatid considered restricted to insects, was recently reported to infect a bat. Herein, C. mellificae has been demonstrated to have a wider range of vertebrate hosts and distribution in Brazilian biomes than once thought. Parasites isolated from haemocultures were characterized using V7V8 SSU rDNA and glyceraldehyde 3-phosphate dehydrogenase genes. Coatis (Nasua nasua) in the Cerrado; marmosets (Callithrix sp.) and bats (Carollia perspicillata, Myotis lavali, M. izecksohni, Artibeus lituratus) in the Atlantic Forest; crab-eating foxes (Cerdocyon thous) and ocelot (Leopardus pardalis) in the Pantanal biomes were infected by trypanosomatids that displayed choanomastigote forms in haemoculture in Giemsa-stained slide smears. Mo lecular characterization and phylogenetic inference confirmed the infection of C. mellificae in these animals. Moreover, slight differences in C. mellificae sequences were observed. Crithidia mellificae growth curves were counted at 27◦C, 36◦C and 37◦C, and the morphotypes were able to grow and survive for up to 16 days. Sero logical titers for C. mellificae were observed in nonhuman primates, demonstrating that this parasite is able to induce a humoral immune response in an infected mammal. These results showed that host specificity in try panosomatids is complex and far from understood.