Abstract Tropospheric ozone (O3) is a major air pollutant that decreases yield of important crops worldwide. Despite long-lasting research of its negative effects on plants, there are many gaps in our knowledge on how plants respond to O3. In this study, we used natural variation in the model plant Arabidopsis (Arabidopsis thaliana) to characterize molecular and physiological mechanisms underlying O3 sensitivity. A key parameter in models for O3 damage is stomatal uptake. Here we show that the extent of O3 damage in the sensitive Arabidopsis accession Shahdara (Sha) does not correspond with O3 uptake, pointing toward stomata-independent mechanisms for the development of O3 damage. We compared tolerant (Col-0) versus sensitive accessions (Sha, Cvi-0) in assays related to photosynthesis, cell death, antioxidants, and transcriptional regulation. Acute O3 exposure increased cell death, development of lesions in the leaves, and decreased photosynthesis in sensitive accessions. In both Sha and Cvi-0, O3-induced lesions were associated with decreased maximal chlorophyll fluorescence and low quantum yield of electron transfer from Photosystem II to plastoquinone. However, O3-induced repression of photosynthesis in these two O3-sensitive accessions developed in different ways. We demonstrate that O3 sensitivity in Arabidopsis is influenced by genetic diversity given that Sha and Cvi-0 developed accession-specific transcriptional responses to O3. Our findings advance the understanding of plant responses to O3 and set a framework for future studies to characterize molecular and physiological mechanisms allowing plants to respond to high O3 levels in the atmosphere as a result of high air pollution and climate change.