The food demand for cereals, feedstocks, and biofuels is increasing worldwide due to continuous population growth and changes in life patterns for food consumption. Climate change is further intensifying abiotic stresses and pest pressures, jeopardizing agricultural productivity. To cope with these challenges, the development of “climate-smart” germplasm and strategic use of genetic diversity is essential under rapidly changing climatic conditions. Genetic diversity, encompassing landraces, accessions, cultivated varieties, breeding stocks, and wild species, is vital for developing nutrient rich, high yielding, pest and disease resistant, and abiotic stress-tolerant cultivars. Characterization, involving morphological, biochemical, physiological, and molecular markers, is essential for identifying desirable traits, thereby advancing breeding initiatives. Among these, molecular markers such as simple sequence repeats (SSRs), single nucleotide polymorphisms (SNPs), and amplified fragment length polymorphisms (AFLPs) are pivotal due to their higher variability, reproducibility, and adaptability to automation. These markers support gene tagging, genetic diversity, phylogenetic analysis, heterosis estimation, genotyping, and marker-assisted selection (MAS). The conservation of plant genetic resources, both in-situ and ex-situ is crucial in maintaining biodiversity and supporting future breeding programs. Molecular markers play a pivotal role in conservation by precisely identifying and characterizing genetic material. This chapter emphasizes the integration of genetic and agronomic interventions to meet future food demands and achieve global agricultural resilience and sustainability.