Regarding the antimonial-resistant of spp., understanding of related mechanism is necessary. One of the most important involved molecules is aquaglyceropin1 (AQP1). The aim of this study was molecular analysis of gene from antimonial-resistant clinical isolates and its expression.Overall, 150 patients with cutaneous leishmaniasis referring to the reference laboratories of Yazd and Varzaneh,, located 105km southeast of Isfahan and 240km away from Yazd, were assessed from Jun 2015 to Dec 2017. After sampling, staining was done and evaluated for Leishman by microscope. Samples were collected in RNAlater solution for gene expression analysis in non-healing isolates. DNA extraction was performed from each slide with Leishman body. All patients with . isolates detected by ITS1-PCR-RFLP were followed for finding the resistant isolates, consequence of molecular characterization of using PCR-RFLP. Gene expression of from all resistant isolates was assessed in comparison with the one in a sensitive isolate. Statistical analysis was done using SPSS. The significance level was considered ≤0.05.Five isolates were detected as antimonial resistant. Molecular detection and identification were appeared that all were . The molecular characterization of showed G562A mutation. Gene expression of in resistant isolates showed 1.67 fold higher than the sensitive isolate.We reported a new point mutation of G562A in gene involved in molecular mechanism in resistant isolates.