BACKGROUND: We aimed to evaluate the accuracy of genotyping of species by the spliced leader mini-exon gene. METHODS: Suspected leishmaniasis patients, referred to Masieh Daneshvary Hospital, Tehran, Iran were included from May 2017 to September 2021. The species were genotyped by PCRRFLP based on the SL mini-exon gene and the region of gene and compared with the sequencing results. The expressed metabolites of metacyclic promastigotes were evaluated by Proton nuclear magnetic resonance (H-NMR). RESULTS: Out of 66 suspected cases, 36 (54.4%) were positive for species based on the PCR assays. In 21 (31.8%) cases, promastigotes grew on culture tubes. Based on the RFLP of SL RNA profile, 13 (19.7%) , 9 (13.6%) , 3 (4.5%) , and 8 (12.1%) isolates, isolated from culture media, were identified; however, 3 (4.5%) cases were unidentifiable due to the low number of parasites. Seventeen metabolites were expressed by the metacyclic forms of , and isolates. The top differential metabolites expressed more in were FAD, p-Methoxybenzyl alcohol and S-b-G-5, 5-G-b-S (A = CH2) (<0.005) whereas Veratryl glycerols and D-(+)-Mannose were significantly increased in and Betulin, LTyrosine in (<0.01). CONCLUSION: The invaluable techniques such as sequencing and 1H-NMR confirmed the results of genotyping of species based on the SL mini-exon gene. SL mini exon gene can be used as a diagnostic tool to differentiate various genotypes and detect contamination of culture media with .