| Summary: | Natural populations of <i>Rhanterium eppaposum</i> Oliv. (Arfaj), a perennial forage shrub, have depleted due to unethical human interventions and climate change in Kuwait. Therefore, there is an urgent need to conserve this native plant through the assessment of its genetic diversity and population structure. Genotyping by sequencing (GBS) has recently emerged as a powerful tool for the molecular diversity analysis of higher plants without prior knowledge of their genome. This study represents the first effort in using GBS to discover genome-wide single nucleotide polymorphisms (SNPs) of local <i>Rhanterium</i> plants to assess the genetic diversity present in landraces collected from six different locations in Kuwait. The study generated a novel set of 11,231 single nucleotide polymorphisms (SNPs) and indels (insertions and deletions) in 98 genotypes of <i>Rhanterium</i>. The analysis of molecular variance (AMOVA) revealed ~1.5% variation residing among the six populations, ~5% among the individuals within the population and 93% variation present within the populations (F<sub>ST</sub> = 0.029; <i>p</i> = 0.0). Bayesian and UPGMA analyses identified two admixed clusters of the tested samples; however, the principal coordinates analysis returned the complete population as a single group. Mantel’s test returned a very weak correlation coefficient of r<sup>2</sup> = 0.101 (<i>p</i> = 0.00) between the geographic and genetic distance. These findings are useful for the native species to formulate conservation strategies for its sustainable management and desert rehabilitation.
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