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Three Leishmania strains were isolated from cutaneous and mucosal lesions of a Colombian male. These strains, shown to be phenotypically identical based on isoenzyme polymorphisms and monoclonal antibody reactivity, were identified as Leishmania braziliensis guyanensis. Six clones of the mucosal strain were phenotypically identical to the 3 strains. The clinical presentation of the cutaneous lesions and the geographic origin of the infection were consistent with infection by L. b. guyanensis.