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Molecular karyotypes of Leishmania isolates from patients with cutaneous leishmaniasis in Ecuador were analyzed by pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization. The DNA karyotypes of L. major-like parasites were similar between two human isolates from a lowland coastal and a highland Andean region, but were apparently different from those of eleven World Health Organization reference strains including L. major. The smallest chromosome of 240 kilobases in L. major-like parasites was found to belong to the 715-class of small linear chromosomal DNAs, which have been shown to appear in some lines of Leishmania. Chromosome banding patterns of L. mexicana isolates exhibited a novel, ordered, chromosomal ladder, and were identical among four human isolates and one canine isolate from a restricted geographic region in the Andes. On the other hand, minor chromosome size polymorphisms were observed among three L. panamensis isolates from different endemic regions near the Pacific Coast. Chromosomal locations of dihydrofolate reductase-thymidylate synthetase and P-glycoprotein genes revealed further differences in chromosomal organizations among these Leishmania species in Ecuador. These results indicate that karyotype analysis by PFGE is useful for epidemiologic studies of leishmaniasis in Ecuador.