Trypanosoma cruzi, the etiologic agent of Chagas disease, presents a complex life cycle, cycling between reduviid vectors and wild and domestic mammals. The European rabbit is an introduced species in America, but its role as reservoir in the wild transmission cycle of Chagas disease remains unknown. We used polymerase chain reaction, Southern blot, and hybridization tests to detect infection and characterize genotypes in rabbits from a hyperendemic area of Chagas disease in Chile. Results show 38% of infection with different genotypes. We provide evidence that rabbits are naturally infected with T. cruzi, which may have important epidemiologic consequences for the wild transmission cycle.