L. infantum and L. donovani, distinct species in the L. donovani complex, show high phenotypic and genotypic polymorphism and share molecular traits. Therefore, genotyping by a single molecular target can give uncertain results. This study focuses on genotyping a set of L. donovani complex strains, including 18 zymodemes classified according to Montpellier nomenclature (ZMONs) and different clinical forms, by internal transcribed spacer (ITS), heat shock protein 70 (hsp70), and cysteine proteinase b (cpb) sequencing to evaluate their ability in species discrimination. We found an unexpected L. infantum hsp70 variability, with 8 sequence variants. Cpb-PCR could not distinguish L. donovani complex species, due to a L. infantum intraspecific allelic (cpbEF) polymorphism. By combining ITS-hsp70-cpb sequence variants, we obtained different genotypes. ITS(A)-hsp70inf(2)-cpbE identified 69.9% of L. infantum strains representing 12 ZMONs from Mediterranean visceral and cutaneous cases, ITS(A)-hsp70inf(2)-cpbF identified a non-ZMON-1 cluster. Four genotypes represented ZMON-24: ITS(A, B)-hsp70(Y)-cpbF identified a cutaneous cluster from Italy and North Africa, ITS(A, Lombardi)-hsp70(2)-cpbE identified cutaneous and visceral cases from Mediterranean areas. We believe this study contributes to an overview of L. infantum variant populations, and to the discussion on diagnostic targets, in single or multi -target-based approaches, to identify Leishmania populations in the Mediterranean area.