In Crocodylidae family three trypanosomes species were described, T. grayi in African crocodilian and T. cecili and Trypanosoma sp. in Caimans species from Brazil. T. grayi was transmitted by tsetse flies and the vector of Brazilian caimans trypanosomes is unknown. We characterized first Brazilian trypanosome isolated in spectacled caiman (Caiman crocodilus) from Mato Grosso State in Brazil. Morphological findings in epimastigotes forms from axenic culture showed high similarity with Trypanosoma sp. described in Caiman yacare from Brazilian Pantanal. Phylogenetic studies performed with SSU rDNA and gGAPDH (glyceraldehydes-3-phosphato dehydrogenase glycosomal) clustering in T. grayi Clade and together to genotype Cay 01 from Trypanosoma unnamed species isolated in C. yacare. This is the first isolate of Trypanosoma sp. from C. crocodilus and the phylogenetic position with isolates in C. yacare from Pantanal region and demonstrates the low host specificity of cayman trypanosomes in Brazil.