Parasitological methods are the most specific procedures used for the diagnosis of Leishmania spp. infection, but their limited sensitivity poses a disadvantage and prompts the need for alternatives. The choice of site for sample collection influences diagnostic sensitivity. The combination of an accurate diagnostic method and a technique that allows large-scale field studies is highly desirable to enhance the investigation of Leishmania spp. infection in dogs, especially in endemic regions. The bone marrow is a good target for the detection of Leishmania spp. in dogs. In this context, bone marrow aspiration is rapid and less invasive compared with biopsy procedures, and also enables cell block processing, paraffin wax embedding and the sectioning of samples for further histological and immunohistochemical analyses. The aim of this study was to describe for the first time parasitological methods (immunohistochemistry [IHC] and histopathology) using the cell block technique with bone marrow aspirates for the diagnosis of Leishmania spp. infection in dogs. Bone marrow aspiration was performed in 45 dogs from an area endemic for visceral leishmaniosis for parasitological culture and the cell block technique (histopathology and IHC). Fourteen (31.1%) dogs tested positive for Leishmania spp. by IHC, six (13.3%) by parasitological culture and four (8.9%) by histopathology. Cell block IHC was a useful tool for the diagnosis of canine visceral leishmaniosis. Further studies should be conducted to validate this method for routine epidemiological screening.