American trypanosomiasis is transmitted in nature via a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, or via a domestic cycle where the parasite comes into contact with humans through domiciliated triatomines. The pool of T. cruzi isolates consists of sub-populations presenting a broad genetic diversity. In contrast to the heterogeneity suggested by isoenzyme analysis, PCR amplification of sequences from the 24S alpha rRNA gene and from the non-transcribed spacer of the mini-exon gene indicated dimorphism among T. cruzi isolates, which enabled the definition of two major parasite lineages. In the present study, 157 T. cruzi isolates obtained from humans, triatomines and sylvatic mammalian reservoirs from 12 Brazilian states were analysed by the 24S alpha RNA and mini-exon typing approaches. The stocks were classified into the two proposed lineages and according to the domestic or sylvatic cycle of the parasite. Data presented provide evidence for a strong association of T. cruzi lineage 1 with the domestic cycle, while in the sylvatic cycle both lineages circulate equally. Molecular typing of human parasite isolates from three well-characterised endemic regions of Chagas disease (Minas Gerais, Paraiba and Piaui) and from Amazonas State, where T. cruzi is enzootic, suggests that in some endemic areas in Brazil there is a preferential linkage between both cycles mediated by lineage-1 stocks.