American tegumentary leishmaniasis (ATL) in Peru is mainly associated with Leishmania (Viannia) peruviana and L. (V.) braziliensis. These parasites are genetically related, and their characterization as distinct species is controversial. Despite their genetic similarity, each species is associated with different clinical manifestations of ATL; L. (V.) peruviana causes only cutaneous leishmaniasis, whereas L. (V.) braziliensis can cause both cutaneous and mucocutaneous leishmaniasis. Because the primary cutaneous lesions caused by infection with these species are indistinguishable, it is necessary to develop a suitable method to differentiate them in order to prevent possible metastasis to oropharyngeal mucosa. In the present study, we investigated the proteolytic profile of L. (V.) peruviana and L. (V.) braziliensis isolates from Peru by zymographic analysis in SDS-PAGE copolymerized with gelatin. Enzymes were characterized according to their pH range of activity and sensitivity to distinct peptidase inhibitors. We observed that L. (V.) peruviana isolates displayed three proteolytic bands with molecular masses ranging from 55 to 80 kDa, whereas L. (V.) braziliensis isolates showed six proteolytic activities between 55 and 130 kDa. Using specific inhibitors, we determined that these proteolytic activities are due to metallopeptidases and present optimal activity between the pH range 5.5 and 10.0. Our results suggest that the expression of metallopeptidases in L. (V.) peruviana and L. (V.) braziliensis isolates from Peru is species-specific.