More than 250 strains of Leishmania isolated from different localities and hosts in the New World were analyzed by enzyme electrophoresis, and their electromorphic profiles were compared with 19 reference strains representing most of the described species of this parasite. The 18 enzymic loci analyzed were very polymorphic, and the strains were classified into 44 zymodemes, each grouping strains with the same enzyme profiles. Each zymodeme was considered as an elementary taxon and the phenetic and phylogenetic relationships were determined by agglomerative hierarchical, ordination, and cladistic techniques. The different classification methods produced very similar results. The 44 zymodemes could be clustered into two groups, corresponding to the subgenera Leishmania and Viannia, by the numerical methods. The subgenus Viannia was shown to be monophyletic and could be further divided into species complexes representing L. braziliensis, L. naiffi, and L. guyanensis/L. panamensis/L. shawi, as well as some isolated taxa including L. lainsoni. The subgenus Leishmania, on the other hand, was polyphyletic, with New World isolates related to L. major clustered separately from the L. mexicana species complex. Most of the other zymodemes in this group represented independent taxa. The results confirm Viannia as a valid taxon but suggest that the status of the subgenus Leishmania should be further investigated. Leishmania braziliensis and L. naiffi were shown to be the most polymorphic species, while L. guyanensis, in spite of being the most common species found in this study, was remarkably homogeneous. The only variants were found south of the Amazon river. North of this river, the species was monomorphic.