Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.