Leishmaniasis is a neglected disease endemic in five continents. It is a severe disease that may lead to death, and its early detection is important to avoid severe damage to affected individuals. Molecular methods to detect Leishmania are considered alternatives to overcome the limitations presented by conventional methods. The aim of this study was to develop multiplex PCR systems able to detect small amounts of target DNA of Leishmania infantum and Leishmania braziliensis, and the gene coding for glyceraldehyde-3-phosphate dehydrogenase (G3PD) in mammals, enabling quality evaluation of the sample simultaneously with detection of the specific target. The systems created for G3PD recognition were combined with detection systems for L. infantum and L. braziliensis to compose multiplex PCR systems for visceral (mVL) and cutaneous (mACL) leishmaniasis diagnosis. The multiplex PCR systems developed were assessed in blood samples from five different species of mammal reservoirs involved in the disease cycle in Brazil, and 96 and 52 human samples from patients with suspected visceral leishmaniasis (VL) and cutaneous leishmaniasis (ACL), respectively. Three G3PD detection systems were created (G3PD1, G3PD2 and G3PD3) with different product sizes, G3PD2 was chosen for the formation of multiplex PCR systems. The two multiplex PCR systems (mVL and mACL) were reproducible in all species evaluated. Results of test samples (sensitivity, specificity and efficiency) suggest its use in routine diagnosis, research activities in medicine and veterinary medicine. Additionally, the systems designed to detect the G3PD gene are capable of combining with other targets used for molecular diagnosis of infectious diseases. Concerning leishmaniasis, the multiplex PCR systems can be used in epidemiological studies for the detection of new and classic reservoirs, which may contribute to the reliability of results and development of actions to control the disease.