The Cellulose synthase-like (Csl) F family has been considered as one of the most crucial genes regulating β-glucan synthesis. It is a cereal cell wall component, holding advantages for human nutrition, but disadvantages in animal nutrition, malting and brewing industries. Based on a genome-wide search method, present study identified barley CslF gene family members by considering the importance of (1,3;1,4)-β-D-glucan and newly completed barley genome. A sum of Eighteen CslF genes were recognized in the barley genome. Then, phylogenetic analyses classified them into 3 groups, that shared conserved motif compositions. A new motif, D,D,WQxxD was also found, which was responsible for the cellulose synthase. Furthermore, using RNA-seq data, the HvCslFs expression profiles were systematically examined in different tissues and tissue-specific candidates were found. Lastly, interaction network analysis identified 11 CslF genes involved in the interaction network. All together, prsent task provides valuable evidence about the genomic organization and evolutionary relationship of the CslF gene family in barley, and facilitate the functional surveys of CslF genes in barley and beyond.