Genotyping-by-sequencing (GBS)-derived molecular markers reveal the distinct genetic population structure and relatively narrow genetic diversity of Chinese hulless oat landraces. Four markers linked to the naked grain gene (N1) are identified by genome-wide association study (GWAS). Interest in hulless oat (Avena sativa ssp. nuda), a variant of common oat (A. sativa) domesticated in Western Asia, has increased in recent years due to its free-threshing attribute and its domestication history. However, the genetic diversity and population structure of hulless oat, as well as the genetic mechanism of hullessness, are poorly understood. In this study, the genetic diversity and population structure of a worldwide sample of 805 oat lines including 186 hulless oats were investigated using genotyping-by-sequencing. Population structure analyses showed a strong genetic differentiation between hulless landraces vs other oat lines, including the modern hulless cultivars. The distinct subpopulation stratification of hulless landraces and their low genetic diversity suggests that a domestication bottleneck existed in hulless landraces. Additionally, low genetic diversity within European oats and strong differentiation between the spring oats and southern origin oat lines revealed by previous studies were also observed in this study. Genomic regions contributing to these genetic differentiations suggest that genetic loci related to growth habit and stress resistance may have been under intense selection, rather than the hulless-related genomic regions. Genome-wide association analysis detected four markers that were highly associated with hullessness. Three of these were mapped on linkage group Mrg21 at a genetic position between 195.7 and 212.1 cM, providing robust evidence that the dominant N1 locus located on Mrg21 is the single major factor controlling this trait.