The Hardness (Ha) locus contains the Hina, Hinb‐1, Hinb‐2, and Gsp genes and was shown to be associated with grain hardness and dry matter digestibility (DMD) variation. In this study, 73 spring barley (Hordeum vulgare L.) accessions selected for DMD variation were assessed for variation in DMD, seed quality, and Hardness (Ha) locus component gene alleles. To determine whether Ha is associated with grain quality traits, we assessed the relationship of the Ha locus in the presence or absence of head type (two or six row) variation. To accomplish this, the barley Ha locus component genes (Hina, Hinb‐1, Hinb‐2, and Gsp) were sequenced from each accession and sorted by prevalence. The most common Ha haplotype (HINA, HINB‐1, HINB‐2 alleles) was present in 42 accessions with the remaining 39 dispersed over 24 haplotypes. Seeds from two‐row accessions with the most common Ha haplotype were significantly softer in grain texture (P < 0.001) and had increased starch content (P < 0.001) and DMD (P < 0.05). We used quantitative reverse‐transcriptase polymerase chain reaction to measure Hina, Hinb‐1, Hinb‐2, and Gsp expression levels in developing seeds of 19 Ha locus haplotypes. The expression levels of all four genes were positively correlated with DMD. The results indicate that selection for individual Ha locus haplotypes may be useful in modifying seed size, DMD, and starch content in two‐row barley.