Naked barley (Hordeum vulgare var. nudum) is a staple food crop contributing significantly to the global food security. Understanding genetic diversity will facilitate its effective conservation and utilization. To determine genetic diversity and its distribution within and among varieties, we characterized 30 naked barley varieties representing the traditional, modern, and germplasm-resources-bank gene-pools from Tibet, using the SSR molecular fingerprints. Results demonstrate abundant genetic diversity in Tibetan naked barley varieties, particularly those in the traditional gene-pool that holds much more private alleles. Principal coordinates and STRUCTURE analyses indicate substantial deviation of the modern varieties from the traditional and germplasm-resources-bank varieties. A considerable amount of seed mixture is detected in the modern varieties, suggesting the practices of using mixed seeds in modern-variety cultivation. Cluster analyses indicate the narrow genetic background of the modern varieties because of a few traditional/germplasm-resources-bank varieties used in breeding. Relationships between variation of genetic diversity and increases in sample sizes within naked barley varieties suggest effective sampling strategies for field collections. Knowledge generated from this study has important implications for the sustainable utilization and effective conservation of different types of naked barley germplasm in Tibet and in other regions of the world.