Leishmania infantum and Leishmania donovani both pertain to the L. (L.) donovani complex and are responsible for visceral leishmaniasis. To explore the L. donovani complex, we focused our study on cysteine protease B (cpb) and especially on 2 cpb copies: cpbE and cpbF. We selected cpb genes because of their phylogenetic interest and host–parasite interaction involvement. Sequencing these 2 copies revealed (i) that cpbE is specific to L. infantum and cpbF is specific to L. donovani and (ii) that these 2 copies are different in length and sequence.Nucleotide sequence data reported in this paper are available in the GenBank database under Accession numbers AY896776 AY896777, AY896778, AY896779, AY896780, AY896781, AY896782, AY896783, AY896784, AY896785, AY896786, AY896787, AY896788, AY896789, AY896790, AY896791. Phylogenetic analysis and protein predictions were carried out in order to compare these copies (i) with other trypanosomatid cpb, especially L. mexicana, and (ii) within the L. donovani complex. Our results revealed patterns specific to the L. donovani complex such as the COOH-terminal extension, potential epitopes and N-glycosylation sites. Moreover, phylogenetic analysis revealed different levels of polymorphism between L. infantum and L. donovani and confirmed the ancestral status of the latter. L. infantum has a shorter sequence and a deleted sequence responsible for modifications in protein conformation and catalytic triad. Considering the clinical aspect, L. infantum dermotropic strains appeared more polymorphic than L. infantum viscerotropic strains.