Fifty-eight stocks of Trypanosoma cruzi from Latin America were genetically characterized using the methods of the polymerase chain reaction (PCR) and the single-stranded conformation DNA polymorphism (SSCP) with four genes, mini-exon, 24Salpha rRNA, 18Sr RNA, cruzipain, and a RAPD fragment DNA region, P7-P8. All the isolates examined were assigned to T. cruzi I or subgroups of T. cruzi II by these methods. From these results, the SSCP analysis, which was simple to perform and highly sensitive to sequence variation, seemed to be a good modality for characterizing T. cruzi, particularly for subgroups of T. cruzi II. However, in several isolates of T. cruzi II, the subgroups determined with the SSCP of 24Salpha rRNA were not consistent with those determined with other genes, the SSCP of 18S rRNA and cruzipain, and the PCR of P7-P8, possibly because of the occurrence of rare genetic exchanges or mutations or both in natural populations of this parasite. The SSCP patterns of 24Salpha rRNA and 18S rRNA were highly variable in the T. cruzi I isolates; therefore, analyses using both genes are considered to be one possible method for the characterization of isolates within T. cruzi I.