The etiological agent of Chagas disease, Trypanosoma cruzi, is subdivided into seven genetic subdivisions termed discrete typing units (DTUs), TcI-TcVI and Tcbat. The relevance of T. cruzi genetic diversity to the variable clinical course of the disease, virulence, pathogenicity, drug resistance, transmission cycles and ecological distribution justifies the concerted efforts towards understanding the population structure of T. cruzi strains. In this study, we introduce a novel approach termed 'phyloquant' to infer the evolutionary relationships and assignment of T. cruzi strains to their DTUs based on differential protein expression profiles evidenced by bottom up large scale mass spectrometry-based quantitative proteomic features. Mass spectrometry features analyzed using parsimony (MS1, iBAQ and LFQ) showed a close correlation between protein expression and T. cruzi DTUs and closely related trypanosome species. Although alternative topologies with minor differences between the three MS features analyzed were demonstrated, we show congruence to well accepted evolutionary relationships of T. cruzi DTUs; in all analyses TcI and Tcbat were sister groups, and the parental nature of genotype TcII and the hybrid genotypes TcV/TcVI were corroborated. Character mapping of genetic distance matrices based on phylogenetics and phyloquant clustering showed statistically significant correlations. We propose the first quantitative shotgun proteomics approach as a complement strategy to the genetic-based assignment of T. cruzi strains to DTUs and evolutionary inferences. Moreover, this approach allows for the identification of differentially regulated and strain/DTU/species-specific proteins, with potential application in the identification of strain/DTU specific biomarkers and candidate therapeutic targets. In