Introduction: During a diagnostic evaluation of canine visceral leishmaniasis (VL), two of seventeen dogs were found to be co-infected by Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi. Methods: Specifi c polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR) assays were performed. Results: PCR assays for Leishmania subgenus identifi cation followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confi rmed the presence of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in those fragments. Conclusions: This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.