Canine leishmaniosis is a widespread disease caused by Leishmania parasites, which are transmitted by phlebotomine sand flies. However, in some areas where canine leishmaniosis is endemic, but the primary vectors have not been found, ticks have been suspected to play a role in transmitting the infection. Herewith, we report the detection of Leishmania infantum kinetoplast minicircle DNA (kDNA) in ticks collected from naturally infected dogs living in rural areas of Southern Italy (site A) and Northeastern Brazil (site B). Between March and October 2007, ticks were collected from 26 dogs positive to anti-Leishmania antibodies (one from site A and 25 from site B) and either placed directly into vials containing 70% ethanol or maintained alive for identification and subsequent dissection. All the 95 ticks collected were morphologically identified as Rhipicephalus sanguineus. After identification, their genomic DNA was extracted (either individually or in pools) and processed by polymerase chain reaction (PCR) for the detection of L. infantum kDNA. Two pools of salivary glands from ticks (one from five females and other from five males) found on a dog from site A and tested by a conventional PCR were positive. Amplicon sequencing confirmed the identity of the parasite. In addition, nine (12.3%) out of the 73 ticks found on dogs from site B and tested by a real-time PCR were positive, with a low parasite load (less than 1 parasite/ml). The retrieval of L. infantum kDNA in salivary glands of R. sanguineus ticks has been here reported for the first time. Therefore, further studies are needed to assess the competence of ticks as vectors of Leishmania parasites from dog to dog.