In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare ), major storage proteins are alcohol‐soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. 65Zn overlay and blotting techniques have been widely used for detecting zinc‐binding protein. However, to our knowledge so far this zinc blotting assay has never been applied to detect a prolamin fraction in barley grains. A radioactive zinc (65ZnCl2) blotting technique was optimized to detect zinc‐binding prolamins, followed by development of an easy‐to‐follow nonradioactive colorimetric zinc blotting method with a zinc‐sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS‐PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc‐binding specificity of certain proteins was detected either by autoradiography or color formation. The dithizone staining method gave similar reproducibility to the radioactive blotting. The detected zinc‐binding protein was identified as B‐hordein by Western blotting.