Human leishmaniasis is highly endemic in Bolivia and shows a growing incidence. This report reveals the genetic variability of 35 isolates mainly belonging to Leishmania braziliensis and Leishmania amazonensis species. Among them, 31 were from human patients with different clinical presentations, 3 strains from Lutzomya nuneztovari anglesi (the proven vector of L. amazonensis) and 1 strain of a mammal (Conepatus chinga). The isolates were analyzed by isoenzyme electrophoresis (MLEE) and PCR-RFLP of ITS rRNA genes, a genetic marker highly polymorphic and better adapted to sub-structuring of populations. MLEE and RFLP-ITS were in agreement to discriminate the species, 12 belong to L. (V.) braziliensis, 21 to L. (L.) amazonensis, 1 to Leishmania (V.) lainsoni and 1 to Leishmania (L.) chagasi. Among L. (V.) braziliensis the RFLP-ITS only highlights variability. Ten isolates from either cutaneous or mucocutaneous clinical forms, were grouped together (bootstrap value of 99.8%) apart from two others, one from a mammal (C. chinga), the other from a patient with a cutaneous form. Among L. (L.) amazonensis both markers detect variability but no significant sub-division was identified including isolates from different clinical forms. Moreover, the high frequency of several isolates from cutaneous forms occurred during an outbreak, with putative hybrid character (multiloci heterozygous patterns depicted by MLEE) could be linked to better fitness of these parasites. However, in the absence of observation of hypothetical parents, their hybrid status remains a question.