This research provided the first view of metabolic and physiological effect of a tissue-specific glaucousness inhibitor in wheat and laid foundation for map-based cloning of the Iw3 locus. Cuticular wax constitutes the outermost layer of plant skin, and its composition greatly impacts plant appearance and plant-environment interaction. Epicuticular wax in the upper part of adult wheat plants can form the glaucousness, which is associated with drought tolerance. In this research, we characterized a glume-specific glaucousness inhibitor, Iw3, by fine mapping, physiological, and molecular approaches. Iw3 inhibits glaucousness formation by altering wax composition. Compared to the wild type, Iw3 eliminated β-diketone, reduced 47 % primary alcohols, but increased aldehyde 400-fold and alkanes fivefold, which led to 30 % reduction of total glume wax load. Loss of the glaucousness increased cuticle permeability, suggesting an important role in drought sensitivity. Genetically, the glaucousness-inhibiting effect by Iw3 is partially dominant in a dosage-dependent manner. We localized the Iw3 locus within a 0.13-cM interval delimited by marker loci Xpsp3000 and XWL3096. Of the 53 wax genes assayed, we detected transcription changes in nine genes by Iw3, downregulation of Cer4-1 and upregulation of other five Cer4 and three KCS homologs. All these results provided initial insights into Iw3-mediated regulation of wax metabolism and paved way for in-depth characterization of the Iw3 locus and the glaucousness-related β-diketone pathway.