Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb-e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb-e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb-e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.