The aim of this experiment was to investigate how two wheat varieties differing in carotenoid concentrations (Pexeso vs. Tercie) affect performance traits, nutrient retention, and meat quality in broiler chickens. A total of 180 one-day-old Ross 308 broiler chicks were randomly allocated to two dietary treatments, each with six replicate pens (15 chicks per pen). A new biofortified spring wheat variety, Pexeso, with increased lutein and zeaxanthin concentrations, was compared with the common Tercie wheat. Both wheat varieties were bred in the Czech Republic by Selgen, a.s. (Prague, Czech Republic). Rapeseed oil served as the primary dietary fat source. The diets were pelleted. Feed and water were provided ad libitum. All birds were weighed on days 0, 14, 28, and 35, and feed intake was monitored. At 28 days of age, six chickens (one per pen; n = 6) with average body weight were selected from each dietary treatment and individually placed in cages to determine the retention of crude protein, fat, tocopherols, and carotenoids. During the balance period, daily feed intake and faeces output were recorded for seven days. At 35 days of age, one chicken with an average body weight was selected from each pen (n = 6) and slaughtered at a commercial slaughterhouse. The breast muscles (pectoralis major) and livers were subsequently dissected for further analyses. Experimental data were statistically analysed using SAS software. Pexeso wheat, with higher carotenoid concentrations, did not affect the body weight of the chickens at 35 days of age or feed intake, but it significantly (p < 0.001) reduced feed conversion ratio. This result was consistent with significantly higher protein retention in chickens (p = 0.004). Higher concentrations of γ-tocopherol (p = 0.006) and lutein (p = 0.004) in the breast muscle, as well as γ-tocopherol (p = 0.047), lutein (p < 0.001), and zeaxanthin (p < 0.001) in the liver, were observed in chickens fed the diet containing Pexeso wheat, compared to Tercie wheat. These findings were supported by higher retention of γ-tocopherol (p = 0.008), lutein (p < 0.001), and zeaxanthin (p < 0.001) resulting from this dietary treatment. In conclusion, Pexeso wheat resulted in greater retention of biologically active antioxidants compared to Tercie wheat, which was reflected in improved performance and meat quality in broiler chickens. This research was funded by the Ministry of Agriculture of the Czech Republic, institutional support MZE-RO0723.