The aim of the experimental part of work was to evaluate the effect of biofortified wheat variety PEXESO with increased content of natural carotenoids in the diet of laying hens on performance characteristics and egg quality. The experiment was carried out in the Institute of Animal Science in Prague Uhříněves. The experiment included 240 laying hens of the Lohmann Brown genotype at the age of 42 weeks. These layers were housed in enriched cages and divided into 4 groups according to wheat variety (TERCIE × PEXESO) and fat source (rapeseed oil × pork lard) in the diet. There were 6 repetitions of ten hens in each group. The experiment lasted 10 weeks. The results were statistically evaluated by SAS software. The addition of PEXESO resulted in an improvement in performance parameters, specifically, it reduced feed intake (P < 0.001) and feed conversion (P = 0.013). Laying hens whose diets included PEXESO produced eggs with reduced Haugh units (P < 0.001), but on the other hand, higher yolk color intensity (P < 0.001) and higher egg shell thickness (P = 0.037) and firmness (P < 0.001).