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Two gene targeted molecular marker systems, CAAT box-derived polymorphism (CBDP) and start codon targeted (SCoT) polymorphism, were used to assess the genetic diversity and relatedness in Prosopis cineraria, a tree of abiotic stress tolerance, agroforestry and ethano-botanical importance. A total of ten wild populations consisting 49 individuals collected from different locations of Indian Thar Desert were examined for the genetic analysis of P. cineraria. Ten CBDP and seven SCoT primers, total 17 primers, generated 204 bands with an average of 12 bands per primer, of which 159 (76.8%) were polymorphic. The average PIC values for both CBDP and SCoT marker were 0.543 and 0.547, respectively. The cumulative data of these two markers were used to analyze different genetic diversity indices and compute pair-wise distances. The population genetic diversity analysis based on cumulative data of CBDP and SCoT markers revealed the high levels of genetic differentiation (GST = 0.341; GST > 0.15 as high), low value of gene flow (Nm = 0.966; Nm > 1 as high) and high fixation index (F-ST = 0. 415). The highest genetic diversity was observed among NGBAR populations followed by CHR populations, while SIK populations showed lowest genetic diversity. AMOVA revealed the percent molecular variation was higher within the populations (77%) compared to that of among populations (23%). The clustering pattern based on UPGMA and PCoA plot clearly demonstrated the genetic relationship among the genotypes collected from the different regions of Indian Thar Desert.