Leishmania infantum and Leishmania donovani both pertain to the L. (L.) donovani complex. The status of certain strains is questioned in the literature and there are no reliable discriminative markers to identify them. Molecular tools are needed to (i) identify diagnostic markers and (ii) to allow a better understanding of phylogenetic relationships. We have developed a PCR based on cysteine protease B (cpb). This PCR discriminates between L. infantum and L. donovani with 50-100pg of DNA. These two species are differentiated by their fragment length. Indeed, L. donovani strains were characterized by a 741-bp product and L. infantum strains by a 702-bp product, except for one strain, which revealed a heterozygous profile with the two products. This PCR does not generate amplification for other Leishmania or kinetoplastids and could contribute to clarify the phylogenetic status of several taxa that are also being debated, such as L. archibaldi.