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Background and Objectives: Leprosy remains an important health problem worldwide. It is one of the oldest recorded diseases of humankind. In this study, we expanded the analysis of the geographic distribution of Mycobacterium leprae by investigating SNPs and rpoT genotypes in South Central Coast and Central Highlands clinical isolates, providing insights into the distribution and transmission of leprosy in Vietnam and in this geographic region., Materials and Methods: 27 clinical isolates from the patients, determined the genotypes of M. leprae by SNP and rpoT polymorphism. SNP genotyping was performed by PCR amplification and sequencing, rpoT genotyping by PCR amplification and electrophoresis., Results: All of 27 DNA samples (100%) were positive with RLEP TaqMan PCR (Ct value range is 18-32 on 3 replicates). SNP type 1 was identified in 15 isolates (56%), while SNP type 3 was detected in 12 samples (44%). SNP type 2 and type 4, were not detected. The 6-base repeat region of the rpoT gene was amplified by PCR and analyzed by 4% MetaPhor™ agarose gel electrophoresis. All isolates yielded amplification products of 91-bp, but not 97-bp., Conclusion: This study showed that 56% of isolates belonged to type 1, 44% to type 3. In addition, all samples have the 3-copy hexamer genotype in the rpoT gene., (Copyright © 2023 The Authors. Published by Tehran University of Medical Sciences.)