In order to investigate the role of potential genotypic differences in three economically important barley cultivars, experiments were carried out to determine the influence of supplemental ultraviolet-B (UV-B, 280–320 nm) radiation on reactive oxygen species (ROS), antioxidant activity and photosynthesis. Greenhouse-grown barley (Hordeum vulgare L.) cultivars ‘Cork’, ‘Prestige’ and ‘Golden Promise’ showed different responses to supplemental 280–320 nm (UV-B) representing 100, 138 and 238% levels of ambient biologically active UV-B radiation, respectively. Among the three cultivars studied, cv. Golden Promise was the most tolerant to UV-B, cv. Prestige was slightly more sensitive than cv. Cork. A comparison with the other two cultivars showed that under supplemental UV-B, Golden Promise leaves (i) retained a higher quantum yield of photosynthesis under photosynthetically active radiation (PAR, 400–700 nm) corresponding to growth conditions; (ii) had the smallest decrease in both electron transport rate and non-photochemical quenching under high PAR; (iii) contained less oxidized ascorbate [measured as dehydroascorbate or electron paramagnetic resonance (EPR) detectable monodehydroascorbate radicals] than either Cork or Prestige. Under the highest UV-B level applied, Golden Promise leaves maintained the same activity of both monodehydroascorbate-reductase (MDAR) and ascorbate-peroxidase (APX) enzymes, as untreated controls, while MDAR markedly decreased in the other two cultivars and APX slightly increased in cv. Prestige. These features, together with the observation of directly EPR-trappable free radicals and the light-independent accumulation of monodehydroascorbate radicals in Cork and Prestige but not in Golden Promise leaves under high UV-B suggest that Golden Promise plants suffered less oxidative stress than the two other cultivars.