Introduction: Trypanosoma cruzi is the etiologic agent of Chagas disease in humans, mainly in Latin America. Trypanosome stocks were isolated by hemoculture from patients followed at Evandro Chagas National Institute of Infectious Diseases (FIOCRUZ) and studied using different approaches. Methods: For species and genotype identification, the stocks were analyzed by parasitological techniques, polymerase chain reaction assays targeted to specific DNA sequences, isoenzyme patterns, besides sequencing of a polymorphic locus of TcSC5D gene (one stock). Results: The isolates presented typical T. cruzi morphology and usually grew well in routine culture media. Metacyclic trypomastigotes were found in cultures or experimentally infected Triatoma infestans. All isolates were pure T. cruzi cultures, presenting typical 330-bp products from kinetoplast DNA minicircles, and 250 or 200-bp amplicons from the mini-exon non-transcribed spacer. Their genetic type assignment was resolved by their isoenzyme profiles. The finding of TcI in one asymptomatic patient from Paraíba was confirmed by the sequencing assay. TcVI was found in two asymptomatic individuals from Bahia and Rio Grande do Sul. TcII was identified in six patients from Pernambuco, Bahia and Minas Gerais, who presented different clinical forms: cardiac (2), digestive with megaesophagus (1), and indeterminate (3). Conclusions: The main T. cruzi genotypes found in Brazilian chronic patients were identified in this work, including TcI, which is less frequent and usually causes asymptomatic disease, unlike that in other American countries. This study emphasizes the importance of T. cruzi genotyping for possible correlations between the parasite and patient’ responses to therapeutic treatment or disease clinical manifestations.