Background: The evolutionary history and ecological associations of Trypanosoma cruzi, the need to identify genetic markers that can distinguish parasite subpopulations, and understanding the parasite’s evolutionary and selective processes have been the subject of a significant number of publications since 1998, the year when the first DNA sequence analysis for the species was published. Methods: The current analysis systematizes and re-analyzes this original research, focusing on critical methodological and analytical variables and results that have given rise to interpretations of putative patterns of genetic diversity and diversification of T. cruzi lineages, discrete typing units (DTUs), and populations, and their associations with hosts, vectors, and geographical distribution that have been interpreted as evidence for parasite subpopulation specificities. Results: Few studies use hypothesis-driven or quantitative analysis for T. cruzi phylogeny (16/58 studies) or phylogeography (10/13). Among these, only one phylogenetic and five phylogeographic studies analyzed molecular markers directly from tissues (i.e. not from isolates). Analysis of T. cruzi DTU or lineage niche and its geographical projection demonstrate extensive sympatry among all clades across the continent and no significant niche differences among DTUs. DTU beta-diversity was high, indicating diverse host assemblages across regions, while host dissimilarity was principally due to host species turnover and to a much lesser degree to nestedness. DTU-host order specificities appear related to trophic or microenvironmental interactions. Conclusions: More rigorous study designs and analyses will be required to discern evolutionary processes and the impact of landscape modification on population dynamics and risk for T. cruzi transmission to humans.