Increasing ambient ozone (O3) concentrations and resurgent rust diseases are two concomitant limiting factors to wheat production worldwide. Breeding resilient wheat cultivars bearing rust resistance and O3 tolerance while maintaining high yield is critical for global food security. This study aims at identifying ozone tolerance among key rust-susceptible wheat genotypes [Rust near-universal susceptible genotypes (RnUS)], as a first step towards achieving this goal. Tested RnUS included seven bread wheat genotypes (Chinese Spring, Line E, Little Club, LMPG 6, McNair 701, Morocco and Thatcher), and one durum wheat line (Rusty). Plants were treated with five O3 concentrations (CF, 50, 70, 90, and 110 ppb), in two O3 exposure systems [continuous stirred tank reactors (CSTR) and outdoor-plant environment chambers (OPEC)], at 21–23 Zadoks decimal growth stage. Visible injury and biomass accumulation rate were used to assess O3 responses. Visible injury data showed consistent order of genotype sensitivity (Thatcher, LMPG 6 > McNair 701, Rusty > Line E, Morocco, Little Club > Chinese Spring). Additionally, leaves at different orders showed differential O3 responses. Biomass accumulation under O3 stress showed similar results for the bread wheat genotypes. However, the durum wheat line “Rusty” had the most O3-sensitive biomass production, providing a contrasting O3 response to the tolerance reported in durum wheat. Chinese Spring was the most tolerant genotype based on both parameters and could be used as a source for O3 tolerance, while sensitive genotypes could be used as sensitive parents in mapping O3 tolerance in bread wheat. The suitability of visible symptoms and biomass responses in high-throughput screening of wheat for O3 tolerance was discussed. The results presented in this research could assist in developing future approaches to accelerate breeding wheat for O3 tolerance using existing breeding materials.