**Abstract**

*Trypanosoma cruzi*, the etiological agent of Chagas disease, presents considerable heterogeneity among populations of isolates within the sylvatic and domestic cycle. This study aims to evaluate the genetic diversity of 14 isolates collected from specimens of *Triatoma vitticeps* from Triunfo, Conceição de Macabu, and Santa Maria Madalena cities (Rio de Janeiro—Brazil). By using PCR based on the mini-exon gene, all isolates showed a profile characteristic of bands zymodeme III and with a lower intensity characteristic of TcII. To verify possible hybrids among the strains analyzed, the polymorphisms analysis of the MSH2 gene was performed. HhaI restriction enzyme digestion products resulted in characteristic TcII fragments only, demonstrating the absence of hybrids strains. In our attempt to characterize isolation in accordance with the reclassification of *T. cruzi* into six new groups called DTUs ("discrete typing unit"), we genotyped the mitochondrial cytochrome oxidase subunit two gene, ribosomal RNA gen (24Sα rDNA), and the spliced leader intergenic region (SL-IR). This procedure showed that TcII, TcIII, and TcIV are circulating in this area. This highlights the diversity of parasites infecting specimens of *T. vitticeps*, emphasizing the habit of wild type and complexity of the region epidemiological study that presents potential mixed populations.

**Keywords:** molecular biology, natural infection, triatomine, *Trypanosoma cruzi*, heterogeneity
