Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2011/20484-7 and 2015/09080-2) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 473343/2012-6), Brazil. SRBU is the recipient of a senior researcher scholarship from CNPq. ACC and CRE were fellows supported by FAPESP (2012/14629-5 and 2016/23405-4).Hsp70 is a cytoplasmic heat-shock protein, encoded by a multicopy tandemly repeated gene that has recently been gaining popularity as a valuable marker for typing Leishmania species. In this study, we used a previously described hsp70 PCR-RFLP method for identifying Brazilian Leishmania isolates. We identified two distinct L. (L.) amazonensis hsp70 alleles that resulted in two different RFLP patterns. Also, we found RFLP polymorphisms amongst L. (Viannia) naiffi strains. The profiles of both L. (V.) shawi and L. (V.) lindenbergi were very similar to those of other L. (Viannia) species. The observations described herein reflect the polymorphism found within species of Leishmania and indicate that results from this hsp70 PCR-RFLP method should be used with caution when typing isolates from clinical cases of leishmaniasis and Leishmania species from Brazil.