Visceral leishmaniasis (VL) has expanded worldwide, and, in Brazil, it has shown growing incidence in urban areas. Serological diagnosis is the main diagnostic method used to detect Leishmania infantum infection in Brazil. The diagnostic protocol recommended by the Brazilian Ministry of Health is a screening assay using a rapid test in dogs (the main urban reservoir of the disease) and humans, followed by a confirmatory ELISA test for dogs and an immunofluorescence assay for humans. Despite the low cost and the ease of performance, the diagnostic tests recommended for VL diagnosis present accuracy problems and cross-reactivity with other species of the Trypanosomatidae family. Therefore, more immunogenic and specific antigens should be selected to avoid cross-reactivity and to enable the detection of more seropositive patients. Therefore, to improve the accuracy of diagnostic tests, our group identified three recombinant proteins of L. infantum (rLci1a, rLci2b, and rLci5) in ELISA and rapid tests, which presented higher sensitivity and specificity than the tests recommended by the Brazilian Ministry of Health. The use of these proteins also showed high accuracy values (84% - 92%). However, there was cross-reactivity with T. cruzi (35% for rLci5), Babesia sp. or Ehrlichia sp. (27% for rLci1, 9% rLci2, and 18% for rLci5). Thus, to improve the accuracy of these proteins in VL diagnosis, bioinformatics analyzes (BLAST-p, Clustal Omega) and peptide microarray studies were carried out in order to identify exclusive and cross- reactive epitopes in L. infantum, T. cruzi, Ehrlichia canis and Babesia canis (species commonly found in coinfections), and select those immunogenic only for Leishmania for use in a diagnostic test. After in silico evaluation, similarity matrices were constructed, high similarities with T. cruzi (92%), E. canis (47%), and Babesia species (64%-74%) were identified for rLc1a. While rLci5 and rLci2b revealed only similarities to proteins within the Leishmania genus. An ELISA was conducted with proteins in their original conformation and after denaturation at 80ºC for 20 minutes to estimate the reactivity to linear epitopes of each protein. Protein rLci2 presented 86% sensitivity, 86% specificity, and 64%, 87%, and 0% cross-reactivity to B. canis, E. canis, and T. cruzi, respectively. Denaturated rLci2 presented the same sensitivity, higher specificity (100%), and less cross-reactivity (45%, 75%, and 0% to B. canis, E. canis, and T. cruzi, respectively). Protein rLci5 presented 86% of sensitivity, 100% of specificity, and 45%, 67%, and 0% of cross-reactivity to B. canis, E. canis, and T. cruzi. Denaturated rLci5 presented lower sensitivity (43%), same specificity, and lower cross- reactivity (27%, 50%, and 0% specificity to B. canis, E. canis, and T. cruzi, respectively). In addition, linear epitopes recognized by anti-Leishmania antibodies were mapped using peptide microarray assays. Seven regions of interest from all three proteins were identified. Candidate epitopes for an accurate diagnosis can be identified from these regions in order to improve the specificity and maintain or improve the sensitivity of VL diagnosis.