The Leishmania genus comprises up to 35 species, of which 20 are responsible for human disease. However, the taxonomic status for many of them is under discussion. The small Heat Shock Proteins (sHSPs) are physiologically relevant, protecting cellular proteins from aggregation and maintaining cellular viability under intensive stress conditions. In Leishmania, a protein of this class was previously described, the 20-kDa heat-shock protein (HSP20), which is encoded by a single gene. In the present study, we used this target, alone or in combination with hsp70 gene, to investigate the phylogenetic relationships among Leishmania species. Using a pair of degenerate primers it was possible amplifying a 370bp fragment of the hsp20 coding region in 39 strains of very different geographic origins, representing in total 16 Leishmania species (14 if L. chagasi and L. archibaldi are considered synonymous names of L. infantum and L. donovani, respectively). Nucleotide sequences were readily obtained by direct sequencing of the amplification products. Both phylogenetic trees and networks based on either hsp20 sequences or combined datasets of hsp20 and hsp70 sequences were constructed. These phylogenic analyses supported the division of the Leishmania genus into nine species: L. (L.) donovani, L. (L.) major, L. (L.) tropica, L. (L.) aethiopica, L. (L.) mexicana, L. (V.) lainsoni, L. (V.) naiffi, L. (V.) guyanensis and L. (V.) braziliensis. Additionally, by network analysis, the subspecies L. (L.) donovani infantum and L. (V.) braziliensis peruviana were recognized within the L. (L.) donovani and L. (V.) braziliensis species, respectively. Therefore, hsp20 gene was found to be a suitable molecular marker for Leishmania typing and classification purposes. In addition, this study represents a solid contribution to the objective of establishing a more reliable taxonomy for the genus Leishmania.