Since 2003, when Paul Hebert proposed “the DNA barcoding” to identify species using a short genetic sequence from a standard part of the genome, like supermarket scanner, a number of targets were selected for species identification, such as the mitochondrial gene of cytochrome oxidase (COI), 18S, internal transcribed spacers (ITS) or 28S of ribosomal DNA, or the spliced leader gene etc. Nowadays, a 2 steps universal method for protists is under development, using 18S as a first step and another gene for group specific barcodes. As concerns kinetoplastids, in 1996, Mac Laughlin et al have opened the way to identify them through typing of the ITSs of ribosomal DNA, suggesting that ITS sequences are genus or even species-specific. Indeed, in 2001, we demonstrated that ITS1 sequence and size were conserved but distinct in all livestock pathogenic trypanosomes from Africa. In 2002 and later, pan-trypanosome primers (TRYP1) proved to be not only able to detect and distinguish African trypanosomes but also T. lewisi (a commensal parasite of rats, which is occasionally zoonotic) and even Leishmania species, including L. martiniquensis/siamensis, responsible of autochtonous leishmaniasis in Thailand. Beyond barcoding, this technique allowed species-specific diagnosis of several kinetoplastids through a single PCR, based on the size of the PCR product. However, in some cases, these consensual primers gave size-confusing products in wild rodents, so, for T. lewisi, primers were designed inside the ITS1 to get a species-specific diagnosis in a second step. A nearly perfect fitting of “species” (a concept hard to defined in some Trypanosomatidae) and “ITS1 sequence” (and most often size) has been observed, which makes of ITS1 a providential target for a very simple barcoding of Trypanosomatidae, however, in some cases, genotyping request to go deeper than the species level. For example, the so-called “species” Trypanosoma evansi, appears to be a subspecies of Trypanosoma brucei