Background: Road-killed wild animals have been classified as sentinels for detecting such zoonotic pathogens as Leishmania spp., offering new opportunities for epidemiological studies of this infection.Methods: This study aimed to evaluate the presence of Leishmania spp. and Leishmania chagasi DNA by PCR in tissue samples (lung, liver, spleen, kidney, heart, mesenteric lymph node and adrenal gland) from 70 road-killed wild animals.Results: DNA was detected in tissues of one Cavia aperea (Brazilian guinea pig), five Cerdocyon thous (crab-eating fox), one Dasypus septemcinctus (seven-banded armadillo), two Didelphis albiventris (white-eared opossum), one Hydrochoerus hydrochoeris (capybara), two Myrmecophaga tridactyla (giant anteater), one Procyon cancrivorus (crab-eating raccoon), two Sphiggurus spinosus (porcupine) and one Tamandua tetradactyla (lesser anteater) from different locations in the Central Western part of Sao Paulo state. The Leishmania chagasi DNA were confirmed in mesenteric lymph node of one Cerdocyon thous. Results indicated common infection in wild animals.Conclusions: The approach employed herein proved useful for detecting the environmental occurrence of Leishmania spp. and L. chagasi, as well as determining natural wild reservoirs and contributing to understand the host-parasite interaction. ; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) ; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)