Comparative genomics is a powerful tool that affords detailed study of the genetic and evolutionary basis for aspects of lifecycles and pathologies caused by phylogenetically related pathogens. The reference genome sequences of three trypanosomatids, T. brucei, T. cruzi and L. major, and subsequent addition of multiple Leishmania and Trypanosoma genomes has provided data upon which large-scale investigations delineating the complex systems biology of these human parasites has been built. Here, we compare the annotated genome sequence of T. rangeli strain SC-58 to available genomic sequence and annotation data from related species. We provide analysis of gene content, genome architecture and key characteristics associated with the biology of this non-pathogenic trypanosome. Moreover, we report striking new genomic features of T. rangeli compared with its closest relative, T. cruzi, such as (1) considerably less amplification on the gene copy number within multigene virulence factor families such as MASPs, trans-sialidases and mucins; (2) a reduced repertoire of genes encoding anti-oxidant defense enzymes; and (3) the presence of vestigial orthologs of the RNAi machinery, which are insufficient to constitute a functional pathway. Overall, the genome of T. rangeli provides for a much better understanding of the identity, evolution, regulation and function of trypanosome virulence determinants for both mammalian host and insect vector.