Summary The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer‐za.227 and cer‐ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer‐za.227 and cer‐ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer‐za alleles were generated by genome editing. The CER‐ZA protein was characterized after expression in yeast and Arabidopsis cer4‐3. Cer‐za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl‐CoA reductase (FAR1). The cer‐ye.267 mutation is located to HORVU4Hr1G063420 encoding β‐ketoacyl‐CoA synthase (KAS1) and is allelic to cer‐zh.54. The amounts of intracuticular waxes were strongly decreased in cer‐ye.267. The cuticular water loss and permeability of cer‐za.227 were similar to wild‐type (WT), but were increased in cer‐ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer‐za.227 and cer‐ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.