Forty-one Trypanosoma cruzi stocks isolated from the Chilean vectors Triatoma infestans and Triatoma spinolai were characterized by pulse-field gel electrophoresis and Southern blotting with the cruzipain gene, and by schizodeme analysis of kinetoplast DNA with EcoRI and Msp I. Seven parasite groups were found by molecular karyotype which correlate with schizodeme and multilocus enzyme electrophoresis, supporting the concept of clonal propagation for Trypanosoma cruzi. A predominant T. cruzi stock was isolated from domiciliary T. infestans in several geographical areas of Chile. In contrast, other frequently found genotypes were circulating in the sylvatic and domestic transmission cycles of specific geographical areas. The greatest heterogeneity of T. cruzi stocks was found among sylvatic T. spinolai where at least 4 genotypes were obtained from a sample of 16 T. cruzi stocks.