The amount of (1,3;1,4)-β-D glucan accumulated in barley ( Hordeum vulgare L.) cell walls is an important consideration for grain end-use. One of the major genes responsible for (1,3;1,4)-β-glucan biosynthesis is HvCslF6 , which was analyzed in this study to determine the allelic variation between low (1,3;1,4)-β-glucan (∼3.3%) cultivar CDC Bold and high β-glucan (∼5.2%) line TR251. The CDC Bold HvCslF6 allele showed 16 single nucleotide polymorphisms (SNPs) and two indels when genomic region downstream of the ATG start codon was compared to TR251 allele. Both indels added 16 nucleotides to HvCslF6 first intron of CDC Bold and a single SNP in the third exon altered alanine 590 codon in the CDC Bold sequence to a threonine codon in TR251 allele. Genetic markers were developed for five polymorphic sites and confirmed useful to select low and high β-glucan lines in a previously characterized CDC Bold/TR251 mapping population and a novel F 5 recombinant inbred line (RIL) population derived from a Merit/H93174006 (4.8 and 5.3% (1,3;1,4)-β-glucan) cross. An analysis of parental lines of six populations segregating for β-glucan concentration validated association between the TR251 HvCslF6 haplotype and high (1,3;1,4)-β-glucan concentration in populations showing a (1,3;1,4)-β-glucan quantitative trait locus (QTL) on chromosome 7H.