Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) is a proteomic technique with proven efficiency in the identification of microorganisms, such as bacteria, fungi, and parasites. The present study aimed to evaluate the usefulness of MALDI-TOF MS for the characterization of species circulating in Bolivia using gene sequencing as a reference technique. 55 strains that were isolated from patients with tegumentary leishmaniasis were analyzed. MALDI-TOF MS identified two species of the complex (,  = 26; . outlier,  = 18), one species of the complex (,  = 1), one species of the complex (,  = 2), and two species of the L. mexicana complex (L. amazonensis,  = 5; and ,  = 3). All of the strains were correctly identified at the subgenus, genus, and complex level, but 10 of them (18%) were misidentified as other species within the same complex by the gene sequencing, with 7 of these corresponding to possible hybrids. Thus, one corresponded to , two corresponded to / possible hybrids, two L. amazonensis corresponded to L. mexicana, and three and two L. amazonensis corresponded to L. mexicana/L. amazonensis possible hybrids. Accordingly, MALDI-TOF MS could be used as an alternative to molecular techniques for the identification of spp., as it is low cost, simple to apply, and able to quickly produce results. In Bolivia, its application would allow for the improvement of the management of patient follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis. The objective of the study was to evaluate the usefulness of MALDI-TOF MS for the characterization of species circulating in Bolivia, in comparison with the sequencing of the gene. In our study, all of the isolates could be identified, and no misidentifications were observed at the complex level. Although the equipment implies a high initial investment in our context, MALDI-TOF MS can be used in different areas of microbiology and significantly reduces the cost of testing. Once the parasite culture is obtained, the technique quickly yields information by accessing a free database that is available online. This would allow for the improvement of the management of patients and follow-ups, the updating of the epidemiological data of the species, and a contribution to the control of tegumentary leishmaniasis in Bolivia. Likewise, it can be used to determine a specific treatment to be given, according to the causal species of , when there are protocols in this regard in the area.