| Summary: | This study describes the morphological, biochemical, and molecular differences among <i>Trypanosoma dionisii</i> isolates from hemocultures of hematophagous (<i>Desmodus rotundus</i>; <i>n</i> = 2) and insectivorous (<i>Lonchorhina aurita</i>; <i>n</i> = 1) bats from the Atlantic Rainforest of Rio de Janeiro, Brazil. Fusiform epimastigotes from the hematophagous isolates were elongated, whereas those of the insectivorous isolate were stumpy, reflected in statistically evident differences in the cell body and flagellum lengths. In the hemocultures, a higher percentage of trypomastigote forms (60%) was observed in the hematophagous bat isolates than that in the isolate from the insectivorous bat (4%), which demonstrated globular morphology. Three molecular DNA regions were analyzed: V7V8 (18S rDNA), glycosomal glyceraldehyde 3-phosphate dehydrogenase gene, and mitochondrial cytochrome <i>b</i> gene. The samples were also subjected to multilocus enzyme electrophoresis and random amplified polymorphic DNA analysis. All isolates were identified as <i>T. dionisii</i> by phylogenetic analysis. These sequences were clustered into two separate subgroups with high bootstrap values according to the feeding habits of the bats from which the parasites were isolated. However, other <i>T. dionisii</i> samples from bats with different feeding habits were found in the same branch. These results support the separation of the three isolates into two subgroups, demonstrating that different subpopulations of <i>T. dionisii</i> circulate among bats.
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