Abstract Background Genetic Exchange in Trypanosoma cruzi is controversial not only in relation to its frequency but also in relation to its mechanism. A mechanism of parasexuality has been proposed based on laboratory hybrids, but population genomics strongly suggests meiosis. In addition, mitochondrial introgression has been reported several times in natural isolates although its mechanism is not clear. Moreover, hybrid DTUs (TcV and TcVI) have inherited at least part of the kinetoplastic DNA (kDNA = mitochondrial DNA) from both parents. Methodology/Principal findings In order to address such topics, we sequenced and analyzed fourteen nuclear DNA fragments and three kDNA maxicircle genes in three TcI stocks which are natural clones potentially involved in events of genetic exchange. We also deep-sequenced (a total of 6,146,686 paired-end reads) the hypervariable region of kDNA minicircles (mHVR) in such three strains. In addition, we analyzed the DNA content by flow cytometry to address cell ploidy. We observed that most polymorphic sites in nuclear loci showed a hybrid pattern in one cloned strain and the other two cloned strains were compatible as parental strains (or nearly related to the true parents). The three clones have almost the same ploidy and the DNA content was similar to the reference strain Sylvio (an almost diploid strain). Despite maxicircle genes evolve faster than nuclear housekeeping ones, we did not detect polymorphism in the sequence of three maxicircle genes showing mito-nuclear discordance. In addition, the hybrid stock shared 66% of its mHVR clusters with one putative parental and 47% with the another one. In contrast, the putative parental stocks shared less than 30% of the mHVR clusters among them. Conclusions/significance The results suggest a reductive division, a natural hybridization, biparental inheritance of the minicircles in the hybrid and maxicircle introgression. The models including such phenomena and that would explain the relationships between these three clones are discussed. Author summary Chagas disease, an important public health problem in Latin America, is caused by the parasite Trypanosoma cruzi . Despite it is a widely studied parasite, several questions about the biology of genetic exchange remain. Meiosis has not been yet observed in laboratory, although inferred from population genomic studies. In addition, previous results suggest that the mitochondrial DNA (called kDNA) may be inherited from both parents in hybrids. Here, we analyzed a hybrid strain and the potential parents to address about the mechanisms of genetic exchange at nuclear and mitochondrial level. We observed that the hybrid strain has heterozygous patterns and DNA content compatible with an event of meiosis. In addition, we observed that the evolutionary histories of nuclear DNA and maxicircles (a part of the kDNA) were discordant and the three strains share identical DNA sequences. Mitochondrial introgression of maxicircle DNA from one genotype to another may explain this observation. In addition, we detected that the hybrid strain shared minicircles (another part of the kDNA) with both parental strains. Our results suggest that hybridization implied meiosis and biparental inheritance of the kDNA. Further research is required to address such phenomena in detail.