Dogs play an important role as reservoirs for human visceral leishmaniasis in Brazil. Therefore, according to the Ministry of Health, the local disease control program is based on the elimination of seropositive dogs. Since the disease can adopt a broad spectrum of clinical signs and the laboratory findings are frequently not conclusive, the detection of specific anti-Leishmania circulating antibodies by means of serologic methods is very helpful in the diagnosis of visceral leishmaniasis. However, the specificity of the serological tests may be harmed due to the occurrence of cross-reaction among diseases such as babesiosis. Thus, the aim of this work was to determine the occurrence of cross-reaction between Babesia sp. and Leishmania chagasi antigens, by ELISA, indirect immunofluorescent antibody test (IFAT) and immunochromatographic dipstick test. The ELISA and IFAT assay were performed using L. chagasi promastigote antigen while the immunochromatographic test employed rK39 antigen. The study was carried out with a group of 12 naturally and experimentally Babesia sp. infected dogs. Of the 12 sera samples tested for visceral leishmaniasis, all of them were found to be negative by ELISA, indirect immunofluorescence and immunochromatographic test. According to the results obtained in the present study it was possible to conclude that cross- reaction between Babesia sp. and Leishmania chagasi antigens by the three methods assessed doesn't occur.