The species Leishmania infantum and L. chagasi are considered causal agents of visceral leishmaniasis, a relevant disease due to its zoonosis and severity in humans. Canines are the main reservoirs, hence the need to implement precise diagnoses to identify the Leishmania species involved. The aim of the study was to perform a molecular diagnosis of leishmaniasis (conventional PCR and Sanger sequencing) in 29 tissue samples from canines clinically suspected of leishmaniasis, previously diagnosed by non-molecular methods. The presence of the parasite could be identified by PCR in 9/29 samples (L. infantum, n=4; L. chagasi, n=4; L. braziliensis, n=1), evidencing a predominance of L. infantum and L. chagasi in canines with cutaneous and mucocutaneous leishmaniasis. The PCR allowed to clarify previously negative diagnoses; however, limitations were found, such as the absence of Leishmania DNA...