Background Trypanosoma cruzi , the protozoan causative of Chagas disease, is classified into six main Discrete Typing Units (DTUs): TcI-TcVI. This parasite has around 10 5 copies of the minicircle hypervariable region (mHVR) in their kinetoplastic DNA (kDNA). The genetic diversity of the mHVR is virtually unknown. However, cross-hybridization assays using mHVRs showed hybridization only between isolates belonging to the same genetic group. Nowadays there is no methodologic approach with a good sensibility, specificity and reproducibility for direct typing on biological samples. Due to its high copy number and apparently high diversity, mHVR becomes a good target for typing. Methodology/Principal findings Around 22 million reads, obtained by amplicon sequencing of the mHVR, were analyzed for nine strains belonging to six T . cruzi DTUs. The number and diversity of mHVR clusters was variable among DTUs and even within a DTU. However, strains of the same DTU shared more mHVR clusters than strains of different DTUs and clustered together. In addition, hybrid DTUs (TcV and TcVI) shared similar percentages (1.9–3.4%) of mHVR clusters with their parentals (TcII and TcIII). Conversely, just 0.2% of clusters were shared between TcII and TcIII suggesting biparental inheritance of the kDNA in hybrids. Sequencing at low depth (20,000–40,000 reads) also revealed 95% of the mHVR clusters for each of the analyzed strains. Finally, the method revealed good correlation in cluster identity and abundance between different replications of the experiment (r = 0.999). Conclusions/Significance Our work sheds light on the sequence diversity of mHVRs at intra and inter-DTU level. The mHVR amplicon sequencing workflow described here is a reproducible technique, that allows multiplexed analysis of hundreds of strains and results promissory for direct typing on biological samples in a future. In addition, such approach may help to gain knowledge on the mechanisms of the minicircle evolution and phylogenetic relationships among strains.