Traits related to spike morphology (SM), including grain density (GD), spike length (SL) and awn length (AL), are of central importance in cereal improvement. A recent study based on a two-row landrace of barley, TX9425, detected QTL controlling all of the three traits in a similar region on the long arm of chromosome 3H. To further characterize this chromosomal region, 12 pairs of near isogenic lines (NILs) for GD were generated from two populations between TX9425 and two different commercial cultivars. A population consisting of 1,028 lines segregating primarily for the target region was also developed using materials generated during the production of these NILs. Results from the analysis of the NILs and the NIL-derived population showed that these three traits were likely controlled by a single-locus which was mapped to a 2.84 cM interval between two SSR markers, GBM1495 and HVM33. Across the 12 pairs of NILs, the presence of the 3HL locus increased GD by 53.4 %, reduced SL and AL by 38.8 % and 62.7 %, respectively. In the NIL-derived population, the presence of the 3HL locus increased GD by 64.6 %, reduced SL and AL by 33.7 % and 62.6 %, respectively. An interesting question arising from this research is why some loci such as the one reported here affect several SM-related traits while others appear to affect one of these traits only. The NILs and the NIL-derived population generated in this study will help answer such questions by providing the germplasm to enable cloning and comparative analysis of the genes responsible for these SM-related traits.