The gene HvDEP1, on barley (Hordeum vulgare L.) chromosome 5H, encodes a γ-subunit of the heterotrimeric G-protein complex and was previously determined to be a candidate gene underlying a major quantitative trait locus for grain length. In the present study, we identified a 9 bp indel (insertion–deletion mutation) at position –84 bp from the start codon within a reported upstream open reading frame located in the 5′ UTR (untranslated region) and developed a diagnostic molecular marker. We also identified a 13 bp indel (–514 bp) in linkage disequilibrium that bridges an important regulatory motif. Using a doubled-haploid population and a barley diversity panel, we were able to show that the effects of these indels were environmentally stable and consistently delineated phenotypic groups based on grain length and 1000-grain weight. Genotypes represented by deletions at these two positions relative to the reference cv. Morex had consistently shorter grains, by 3.69–3.96%, and lower 1000-grain weight, by 2.38–4.21%, in a doubled-haploid population studied. Additionally, a diversity panel was tested but consistent differences were observed only for grain length, reinforcing literature indicating the importance of this gene for grain-length regulation. The frequency of the longer and heavier grained reference allele was higher in modern cultivars, suggesting that indirect selection for longer grain may have occurred through direct selection for grain yield via grain-weight improvement. These results indicate that grain length and 1000-grain weight in barley can be manipulated by targeting variation in gene promoters through marker-assisted selection.