Grain shattering in standing crops of wheat (Triticum aestivum L.) is estimated to account for up to 17% loss of harvestable yield. In this study, we analysed data from two bi-parent populations and a wheat diversity panel to explore the underlying genetic basis for grain shattering observed in multiple field experiments through quantitative trait loci (QTL) analysis. Grain shattering had a significant and negative association with grain yield, irrespective of populations and environments. The correlation with plant height was positive in all populations, but correlations with phenology was population-specific, being negative in the diversity panel and the Drysdale × Waagan population, and positive in the Crusader × RT812 population. In the wheat diversity panel, allelic variations at well-known major genes (Rht-B1, Rht-D1 and Ppd-D1) showed minimal association with grain shattering. Instead, the genome-wide analysis identified a single locus on chromosome 2DS, which explained 50% of the phenotypic variation, and mapping to ~10 Mb from Tenacious glume, Tg gene. In the Drysdale × Waagan cross, however, the reduced height (Rht) genes showed major effects on grain shattering. At the Rht-B1 locus, the Rht-B1b allele was associated with 10.4 cm shorter plant height, and 18% decreased grain shattering, whereas Rht-D1b reduced plant height by 11.4 cm and reduced grain shattering by 20%. Ten QTL were detected in the Crusader × RT812, including a major locus detected on the long arm of chromosome 5A. All the QTL identified in this population were non-pleiotropic, as they were still significant even after removing the influence of plant height. These results indicated a complex genetic system for grain shattering in modern wheat, which varied with genetic background, involved pleiotropic as well as independent gene actions, and which might be different from shattering in wild wheat species caused by major domestication genes.