Parasitic protozoa within the taxon Trypanosoma cruzi are considered to be derived from multiple clonal lineages, and show broad genetic diversity as a result of propagation with little or no genetic exchange. We have analyzed a wide sample of T. cruzi isolates from vertebrate and invertebrate hosts by PCR amplification of a ribosomal RNA gene sequence, a mini-exon gene sequence and random amplified polymorphic DNA (RAPD). Amplification of the distinct rDNA and mini-exon gene sequences indicated a dimorphism within both of the tandemly-repeated genes: 125 or 110 bp products for rDNA and 300 or 350 bp products for the mini-exon. Within individual isolates, one of three associations was observed: the 125 bp rDNA product with the 300 bp mini-exon product (defined as group 1), the 110 bp rDNA product with the 350 bp mini-exon product (defined as group 2) and the presence of both rDNA amplification products with the mini-exon group 1 product (group 1/2). The RAPD analysis showed variability between individual isolates, however, tree analysis clearly indicated the presence of two major branches. Interestingly, the rDNA/mini-exon group 2 isolates correlated precisely with one branch of the RAPD-derived tree; group 1 and group 1/2 isolates correlated with the other branch. Our studies show a clear division of T. cruzi into two major lineages presenting a high phylogenetic divergence. Hypotheses are discussed to explain the origin of the two lineages as well as isolates that are hybrid for group 1 and 2 rDNA markers.