Kinetoplast DNA (kDNA) was isolated from 56 stocks of Trypanosoma cruzi isolated from human patients, animals and insects from Brazil, Venezuela, Colombia and Costa Rica. Comparison of the patterns of digested kDNA on acrylamide gels led to the grouping of several stocks into two schizodemes. Schizodeme analysis was also performed using a set of 330-bp fragments representing all the variable regions of the minicircle DNA molecules, which were obtained by PCR amplification of the kDNA using conserved region primers. The results of this analysis were consistent with the analysis using total kDNA, but the more informative restriction profiles allowed the construction of additional schizodemes. In addition, two oligomers were generated from variable region sequences of cloned minicircles from a Y and a CI strain, and these were used as schizodeme-specific probes to detect homologous sequences in the amplified minicircle DNAs. The results indicate that a combination of restriction enzyme fingerprinting and hybridization of amplified variable region minicircle DNA with schizodeme-specific probes can be used for both sensitive detection and classification of T. cruzi.