An important component of primary cell walls in barley (Hordeum vulgare) is (1,3;1,4)-β-glucan, a polymer that has proven health benefits in the human diet, but that is undesirable for the brewing and distilling industry as it influences grain processability. Up to now, the main gene identified as having an involvement in (1,3;1,4)-β-glucan biosynthesis is Cellulose synthase-like F6 (CslF6), but many aspects of the biosynthesis remain unclear. Using the barley Morex genome sequence assembly, new genes of the CslF family (HvCslF11 and HvCslF12) were identified and tested for a potential function in (1,3;1,4)-β-glucan biosynthesis using a transient expression system in Nicotiana benthamiana. This analysis highlighted the importance of HvCslF6 as none of the other proteins were able to synthesis (1,3;1,4)-β-glucan in substantial amounts. For the identification of further genes involved in (1,3;1,4)-β-glucan synthesis, different bioinformatics studies were conducted. To identify genes which could regulate HvCslF6 expression and therefore potentially influence (1,3;1,4)-β-glucan content, an eQTL study was conducted. For the identification of genes possibly involved in the biosynthetic pathway, a co-expression study was undertaken. A combination of different expression datasets from microarray and RNA-seq experiments was used to identify potential candidates. This identified 46 co-expressed candidate genes. From this list three candidates were chosen for functional testing. To investigate the involvement of those genes in (1,3;1,4)-β-glucan biosynthesis knockout plants were created using CRISPR-Cas9 genome editing. As a control HvCslF6 was included in this experiment as well. For three of those four constructs, heterozygous plants for different mutations were generated. A first analysis of (1,3;1,4)-β-glucan content in the T0 resulted in a range from 3 to 5 (% w/w) which is in a normal range for spring barley, but a more detailed phenotypic analysis is planned for plants homozygous for a mutation in the T2.