To expand the available panel of recombinant proteins that can be useful for identifying Leishmania -infected dogs and for diagnosing human visceral leishmaniasis (VL), we selected recombinant antigens from L. infantum , cDNA, and genomic libraries by using pools of serum samples from infected dogs and humans. The selected DNA fragments encoded homologs of a cytoplasmic heat-shock protein 70, a kinesin, a polyubiquitin, and two novel hypothetical proteins. Histidine-tagged recombinant proteins were produced after subcloning these DNA fragments and evaluated by using an enzyme-linked immunosorbent assays with panels of canine and human serum samples. The enzyme-linked immunosorbent assays with different recombinant proteins had different sensitivities (67.4–93.0% and 36.4–97.2%) and specificities (76.1–100% and 90.4–97.3%) when tested with serum samples from Leishmania -infected dogs and human patients with VL. Overall, no single recombinant antigen was sufficient to serodiagnosis all canine or human VL cases.