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This study reports the genetic diversity of Terfezia boudieri collected from southern Tunisia. The study was earned out using 135 truffle fruiting bodies harvested from seven different locations. Twenty-eight Terfezia claveryi fruiting bodies were also sampled from one of the seven locations. A PCR-based technique was used to amplify the internal transcribed spacer (ITS) region of the rDNA, including the ITS1-5.8S-ITS2. The PCR products were digested with the four restriction enzymes Rsal, HhaI, AluI, and HinfI. Based on the Hinfl patterns, T boudieri specimens were separated into two different haplotypes (I and II) Nucleotide sequences of some representative amplicons were also obtained. Based on the phylogenetic results, three T. boudieri genotypes could be differentiated. One sequence, SKtb1, retrieved from PCR-RFLP of haplotype I, was obtained from a low pH soil in association with Helianthemum kahiricum. Based on the results presented in the current study, this isolate may represent a novel taxa within the Terfezia genus.