Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts. The T. rangeli haploid genome is ∼24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heat-shock proteins. Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets. 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.