Background: Although canine visceral leishmaniasis (CVL) transmission primarily occurs through the bite of phlebotomine sand flies infected with Leishmania infantum, alternative routes may exist. Methods: Thirty-four dogs diagnosed with CVL were sampled for parasitological investigation in tissues from the reproductive tract. Results: Amastigotes of Leishmania sp. were present in 79% (27/34) of the reproductive system samples, with distinct infection rates depending on the tissue. Conclusions: Our data confirms that alternative routes, such as horizontal and vertical transmissions, should be considered in the epidemiological chain of CVL.