Barley (Hordeum vulgare L.) is an important cereal crop valued for its nutritional benefits and adaptability to diverse climates. β-glucan, a soluble dietary fiber found in barley, is recognized for its health benefits, including lowering cholesterol, managing postprandial blood glucose levels, and providing antioxidative properties. However, high β-glucan content can complicate food processing due to increased viscosity and water absorption rates. This study used genotyping-by-sequencing (GBS) to examine genetic variation within barley populations and to identify genetic markers associated with β-glucan content. A Genome-Wide Association Study (GWAS) was conducted to identify candidate genes linked to β-glucan levels. Functional annotation revealed several genes potentially involved in cellulose synthase activity and cell wall biosynthesis, including the MYB-related protein Zm38, C2 domain-containing protein, ATP synthase subunit beta (chloroplastic), ATP-dependent Clp protease proteolytic subunit, soluble inorganic pyrophosphatase, and glyceraldehyde-3-phosphate dehydrogenase 1 (cytosolic). These findings provide insights into the genetic architecture of β-glucan content in barley. By leveraging GBS and GWAS, breeders can identify and select genetic markers associated with high β-glucan content, thereby facilitating the development of superior barley varieties with enhanced nutritional and processing qualities.