Trypanosomes isolated from South American bats include the human pathogen Trypanosoma cruzi. Other Trypanosoma spp. that have been found exclusively in bats are not well characterized at the DNA sequence level and we have therefore used the SL RNA gene to differentiate and characterize kinetoplastids isolated from bats in South America. A Trypanosoma sp. isolated from bats in southern Brazil was compared with the geographically diverse isolates T. cruzi marinkellei, T. vespertilionis, and T. dionisii. Analysis of the SL RNA gene repeats revealed size and sequence variability among these bat trypanosomes. We have developed hybridization probes to separate these bat isolates and have analysed the DNA sequence data to estimate their relatedness. A new species, Trypanosoma desterrensis sp. n., is proposed, for which a 5S rRNA gene was also found within the SL RNA repeat.