Visceral Leishmaniasis (VL), also known as kala-azar, is a disseminated protozoan infection caused by the Leishmania donovani complex. Traditionally, the definitive diagnosis is made by detecting amastigotes in biological tissue samples. In August 2015, an apparently healthy, young adult, female, mongrel dog from the Zoonosis Control Center of the Rolim de Moura,Rondônia, was subjected to the canine visceral leishmaniasis diagnosis. The diagnosis was conducted using bone marrow aspirate smears and popliteal lymph node smears by Fine-needle aspiration biopsy (FNAB); stained slides with Diff-Quick screened for Leishmania amastigotes by means of direct optical microscopic examination (100×). Lymph node and bone marrow aspirates were used to investigate the presence of Leishmania infantum chagasi DNA by real-time PCR. The popliteal lymph node positive result was observed in typical amastigotes, presenting nucleus and kinetoplasts associated with lymphoblastic proliferation, reactive macrophages, plasmocytes (commonly Mott cells), eosinophils, and lymphoglandular hyperplasia. A positive bone marrow sample resulted from the observation of amastigotes in the monocyte cytoplasm or free on the smear background. The presence of the L. infantum kDNA was detected in lymph node and bone marrow FNAB samples. This research note describes the autochthonous case of CVL recorded in the state of Rondônia, Brazil, a non-endemic area for VL. According to the data, future studies must include a larger number of animals to elucidate the parasite’s epidemiological resource in Rondônia.