Trypanosoma cruzi, the agent of Chagas disease, has a basically clonal population structure with rare hybridization events. The species is subdivided into six "Discrete Typing Units" called DTUs I, IIa-e, distributed into two major phylogenetic lineages, T. cruzi I and II (TC I and II). The glucose-6-phosphate isomerase (Gpi) is a specific isoenzymic locus that presents homozygous profiles for DTUs I, IIa-c, and typical heterozygous patterns, for DTUs IId and IIe. The gene was sequenced in 12 T. cruzi stocks and in three stocks pertaining to related species. The phylogenetic relationships observed confirm that the DTUs I, IIa-c do constitute monophyletic groups. Nevertheless, the phylogenetic hierarchy of the DTUs is not clearly resolved with the GPI gene. The hybrid status of DTUs IId and IIe was clearly supported. Sequence analysis revealed that the allele 4 present in both DTUs IIa and IIc, previously considered as unique, displayed in fact two distinct sequences, specific for each DTU. The level of recombination between alleles has been investigated.