Fifty-four stocks of Trypanosoma cruzi from vectors, mammalian reservoirs, and infected humans were characterized by enzyme electrophoresis in starch gels using Brazilian zymodeme reference strains (Z1, X-10; Z2, ESM; Z3, CAN-3) as standards. Colombian stocks were collected in three ecologically and epidemiologically distinct settings. Thirteen enzymes were included in the evaluation. Sixteen different phenotypic profiles or “zymodemes” were evident and generated three groups of closely related stocks: a sylvatic Z1-like group, a domiciliary Z1-like group, and a sylvatic Z3-like group. The number of zymodemes observed in foci of sylvatic transmission was greater than in foci of domiciliary transmission. Modified ecologic conditions associated with agriculture and the consequent reduction of biologic diversity may account for the observed pattern of zymodeme distribution and heterogeneity. The phenotypic similarity between the principal sylvatic group of stocks and domiciliary stocks contrasts with the extensive differences observed between the domestic Z2 zymodeme and sylvatic Z1 and Z3 zymodemes in Brazil and Chile.