We ascertain the in vitro Benznidazole (BZN) and Nifurtimox (NFX) susceptibility pattern of epimastigotes, trypomastigotes, and amastigotes of 21 T. cruzi strains, from patients, reservoir and triatomine bugs of various geographic origin. Using this panel of isolates, we compute the Epidemiological cut off value (CO_wt). Then, the frequency of the susceptible phenotype (Wild type) towards BZN and nifurtimox (NFX) within this set of strains belonging to 3 discrete typing units (DTUs), TcI, TcII, and TcV was deduced. We have observed that the susceptibility status of individual T. cruzi isolates toward BZN and NFX is related to the genetic background and to underlying factors probably related to the individual life trait history of each strain. Analyzing drug susceptibility in this conceptual framework would offers the possibility to evidence a link between isolates expressing a low susceptibility level (not wild-type) as define by the CO_wt value and none-curative treatment. It will also permit to tract drug-resistant parasites in T. cruzi population.