Leishmaniasis represents a complex of diseases with a broad clinical spectrum and epidemiological diversity. Involving a variety of agents, reservoirs, and vectors, cutaneous leishmaniasis (CL) presents different transmission patterns, which makes its control especially difficult. Epidemiological characterization with identification of existing Leishmania species in a particular region is essential for understanding this parasitosis epidemiology. Because similar results may be observed in places with overlapping transmission of visceral and cutaneous leishmaniasis, contributing to knowledge about the epidemiological cycle of cutaneous leishmaniasis is of global interest. This study proposes to identify the different species of Leishmania present in human CL cases in the state of Mato Grosso do Sul, Brazil. Slides of imprint tissue were selected from patients diagnosed with CL from 2009 to 2013 at the Laboratório de Parasitologia Clínica, Universidade Federal de Mato Grosso do Sul. The slides were scraped and extracted genetic material was amplified with PCR addressing the Leishmania spp. kDNA region. The reaction product was subjected to RFLP for the specific identification of Leishmania. Forty-three samples (82.7%) amplified with PCR were considered positive. Of these, it was possible to determine the species in 40 samples (76.9%) through analysis of electrophoresis profiles. Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis, and Leishmania (Leishmania) infantum were identified as the etiologic agents of CL in Mato Grosso do Sul. The results of this study permit a more detailed understanding of CL epidemiology and the geographical distribution of its agents.2050-01-01