Dihydrofolate reductase-thymidylate synthase (DHFR-TS) and trypanothione reductase (TR) are important enzymes for the metabolism of protozoan parasites from the family Trypanosomatidae (e.g. Trypanosoma spp., Leishmania spp.) that are targets of current drug-design studies. Very limited information exists on the levels of genetic polymorphism of these enzymes in natural populations of any trypanosomatid parasite. We present results of a survey of nucleotide variation in the genes coding for those enzymes in a large sample of strains from Trypanosoma cruzi, the agent of Chagas' disease. We discuss the results from an evolutionary perspective. A sample of 31 strains show 39 silent and five amino acid polymorphisms in DHFR-TS, and 35 silent and 11 amino acid polymorphisms in TR. No amino acid replacements occur in regions that are important for the enzymatic activity of these proteins, but some polymorphisms occur in sites previously assumed to be invariant. The sequences from both genes cluster in four major groups, a result that is not fully consistent with the current classification of T. cruzi in two major groups of strains. Most polymorphisms correspond to fixed differences among the four sequence groups. Two tests of neutrality show that there is no evidence of adaptive divergence or of selective events having shaped the distribution of polymorphisms and fixed differences in these genes in T. cruzi. However, one nearly significant reduction of variation in the TR sequences from one sequence group suggests a recent selective event at, or close to, that locus.