Forty-three Trypanosoma cruzi isolates from Chile and Colombia and three cloned stocks from Bolivia and Brazil were studied at the karyotype level by hybridization with four different parasite gene probes to chromosomes separated by pulsed-field gel electrophoresis. The results showed that classification of parasite isolates based on isoenzyme analysis at 12 or more genetic loci correlated with the classification obtained by molecular karyotype analysis. However, less correlation was found between molecular karyotypes and the zymodemes Z1, Z2Bra, Z2Bol, and Z3 based on analysis at only two genetic loci. All the four probes used in this study allowed differentiation between different T. cruzi stocks but the SAPA and the antigen 13 probes were most informative. Isolates which were unclassified at the isoenzyme level were also studied and in most cases similar hybridization patterns were observed as obtained with one or more isoenzyme-classified isolates. The results demonstrate the potential of using molecular karyotyping as a tool for classification. A few pulsed-field gel electrophoresis hybridization experiments provide the same information as obtained by isoenzyme analysis using a dozen or more enzymes. The clonal theory of T. cruzi propagation is supported by our results since the strong correlation between isoenzyme classification and molecular karyotype is difficult to explain with a sexual mode of replication. No minichromosomes were detected in any of the T. cruzi samples studied. Neither was any strong correlation found between the clinical manifestations of Chagas' disease and the molecular karyotypes of the T. cruzi isolates.