Leishmaniasis is a disease of public importance with a complex transmission cycle. A quantitative PCR was developed by using the small subunit of the ribosomal RNA gene (SSU rRNA) as a DNA target, which is conserved in all Leishmania species. A TaqMan ® probe was designed to have a high specificity. In all, 22 out of 23 (95.7%) ticks classified as R. microplus tested positive for Leishmania sp. The quantification was between 34.1 and 2197.1 parasites per tick in a range of 12 to 769 fg/uL. In addition, 9 out of 10 (90%) ticks classified as Amblyomma sabanerae tested positive for Leishmania sp. The quantification was between 448.6 and 5428.6 parasites per tick in a range of 157 to 1900 fg/µL. Leishmania sp. was identified in very high percentages in Rhipicephalus microplus and Amblyomma sabanerae from wild Pecari tajacu and Chelonoidis denticulata, in quantities of 34.1 and 5428.6 parasites per arthropod, and this could suggest that the ticks were parasitized by sucking blood from the animals from which they were collected. This is the first report about Leishmania parasites found in wild Rhipicephalus microplus and Amblyomma sabanerae, adding new information about the distribution and epidemiology of the parasite in sylvatic areas.