The purpose of this work was a Canine Visceral Leishmaniasis--CVL study by parasitological direct examination of Leishmania (L.) chagasi (imprinting and histological), immunohistochemical test and histopathological analysis using spleen tissues from 34 dogs euthanized by the Zoonotic Disease Control Centre from Ilha Solteira, SP, Brazil. According to the clinical signs, the dogs were divided in three groups: asymptomatics (8 dogs), oligosymptomatics (17 dogs) and symptomatics (9 dogs). After the accomplishment of all diagnostic tests, 22 dogs were considered positives (64.7%) and 12 (35.3%) were negatives to CVL. From these positive dogs, 1/22 (4.5%) was asymptomatic, 12/22(54.5%) were oligosymptomatics and 8/22 (40.1%) were symptomatics. The histopathological study in spleen tissues from positive, especially symptomatic dogs, showed a diffuse chronic inflammation with thickness of capsular and trabecular regions and there was extensive morphologic alteration of the red and white pulp by the presence of abundant macrophages full with amastigotes, the granulomatous inflammatory reaction and haemorrhagic areas. The data of this work from histopathologic examination and direct microscopic visualization of L. (L.) chagasi showed that the spleen was an useful organ to collect sample tissues for CVL diagnosis. The immunostaining detected the highest number of positive dogs and were considered an important and conclusive method to be used in addition to parasitological methods for CVL, particularly in asymptomatic or oligosymptomatic dogs.