Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.