Naked barley (Hordeum vulgare var. nudum) is a staple food crop, contributing significantly to 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 from Tibet, representing the traditional, modern, and germplasm-resources-bank gene pools, by analyzing SSR molecular fingerprints. The results demonstrate abundant genetic diversity in Tibetan naked barley varieties, particularly those in the traditional gene pool that holds much more private (unique) 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 further indicate the narrow genetic background of the modern varieties, likely due to the limited number of traditional/germplasm-resources-bank varieties applied in breeding. Relationships between increases in genetic diversity and sample sizes within naked barley varieties highlight the importance of effective sampling strategies for field collections. The findings from this study have important implications for the sustainable utilization and effective conservation of different types of naked barley germplasm, both in Tibet and in other regions around the world.