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Four Leishmania sp. samples were isolated from autochthonous human cases of American cutaneous leishmaniasis (ACL) in Santa Catarina State, southern Brazil. These strains were characterized using indirect immunofluorescence with a panel of Leishmania-specific monoclonal antibodies (MAbs), and by PCR amplification and hybridization assay of the mini-exon gene with group specific probes. The results obtained with the MAbs were in agreement with the genetic marker. Two isolates (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were identified as L. (Leishmania) amazonensis and two (MHOM/BR/96/LSC96-H3 and MHOM/BR/97/LSC97-H4) as L. (Viannia) braziliensis. The southernmost autochthonous cases of ACL in Brazil are due to two different Leishmania sp. species, confirming the spreading of ACL on the American continent.