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The plant adaptation response to a changing environment depends on the genetic diversity level it possesses. Genetic diversity and a thorough understanding of population indices are pivotal for decoding plant adaptation to dynamic environmental stressors. The development of polymerase chain reaction (PCR)-based molecular markers enables comprehensive population analyses and the precise detection of individuals and groups with unique genetic variations. Various molecular markers have been employed to assess genetic diversity, examine population structure, and delineate cluster patterns within and among populations. DNA markers revolutionize plant diversity studies by allowing detailed analyses of genetic variations, including economically significant trait-influencing genes. Despite their simplicity, they offer high reproducibility, ensuring accurate estimations of plant variation. Integrating multiple marker systems with advanced high-throughput sequencing techniques is poised to enhance the understanding and management of depleting plant genetic resources by providing a comprehensive picture of diversity at the genome-wide level. This review explores diverse molecular markers, elucidating their advantages and limitations, and highlights their impact on evaluating the genetic diversity and population structure of plants.