BACKGROUND: Cutaneous leishmaniasis caused by species () is one of the most important parasitic diseases in humans. To gain information on the metabolite variations and biochemical pathways between , we used the comparative metabolome of metacyclic promastigotes in the Iranian isolates of and by proton nuclear magnetic resonance (H-NMR). METHODS: and were collected from three areas of Iran, namely Gonbad, Mashhad, and Bam, between 2017 and 2018, and were cultured. The metacyclic promastigote of each species was separated, and cell metabolites were extracted. H-NMR spectroscopy was applied, and the data were processed using ProMatab in MATLAB (version 7.8.0.347). Multivariate statistical analyses, including the principal component analysis and the orthogonal projections to latent structures discriminant analysis, were performed to identify the discriminative metabolites between the two . Metabolites with variable influences in projection values of more than one and a P value of less than 0.05 were marked as significant differences. RESULTS: A set of metabolites were detected, and 24 significantly differentially expressed metabolites were found between the metacyclic forms of and isolates. The top differential metabolites were methionine, aspartate, betaine, and acetylcarnitine, which were increased more in than (P<0.005), whereas asparagine, 3-hydroxybutyrate, L-proline, and kynurenine were increased significantly in (P<0.01). The significantly altered metabolites were involved in eight metabolic pathways. CONCLUSION: Metabolomics, as an invaluable technique, yielded significant metabolites, and their biochemical pathways related to the metacyclic promastigotes of and . The findings offer greater insights into parasite biology and how pathogens adapt to their hosts.