We have developed and tested a new way of typing Trypanosoma cruzi, namely the use of cloned nuclear DNA fragments as genetic markers. Restriction fragment length polymorphisms were verified on Southern blots hybridized to random probes. Fragment patterns were analyzed and dendrograms constructed. Our results on well characterized laboratory strains correlate well to published isoenzyme studies. Some of the probes were also hybridized to chromosomes separated by pulse field gel electrophoresis and a higher degree of heterogeneity was observed at this level.