Our strategy of probes designing for major clones of Trypanosoma cruzi was performed taking into account the: (i) clear identification of the major clones under multilocus study; (ii) hypothesis of a parallel evolution between the extranuclear and nuclear markers; (iii) structure of kDNA which allowed to amplify high variable regions of the minicircle (HVRm) by PCR. The large production of HVRm was very useful to test their ability to be used as probes for detection of DNA from a diversified genetic panel of T. cruzi. Our success in designing such probes has important implications on: the enhancement of 2 evolutive hypothesis about the clonal structure of T. cruzi and the parallel evolution of their extranuclear and nuclear genetics markers; direct diagnosis in patients and vectors. Studies on bio-clinical significance of major clones are discussed. This procedure could be used as a general strategy to generate DNA probes for Kinetoplastida.