Diagnostic tools are important for clinical management and epidemiological evaluation of Tegumentary (TL) and Visceral (VL) Leishmaniasis. Serology is not frequently used for the diagnosis of the TL form because low antibody titers and cross-reaction with VL. Therefore, it is crucial to identify specific and immunogenic antigens from species associated with the TL form. Here we employed a proteomic approach coupled to an in silico analysis and identified the most abundant and immunogenic proteins from Leishmania amazonensis, Leishmania braziliensis and Leishmania infantum. Of 16 species specific proteins, nine were from the species causative of the TL form (L. amazonensis and L. braziliensis). In silico analysis revealed 18 B-cell epitopes with 0% similarity to Trypanosoma cruzi orthologs and, therefore, less likely to crossreact with sera of patients with Chagas disease. Two proteins reacted exclusively with serum from TL patients and presented several B-cell epitopes without similarity to T. cruzi orthologs: the hypothetical protein GI 134063939 and the metallo-peptidase Clan MA(E)-Family M3. The immunoassay revealed nine peptides with strong reactivity to sera from TL patients. These proteins and peptides may be good candidates to improve the specificity and sensibility of serological tests aiming to diagnose the TL of this neglected human disease.