Roots are key targets of ozone (O3) impact in Pima cotton, altering both root and (indirectly) shoot function. O3 reduces phloem loading, biomass allocation to roots, root to shoot biomass ratio, root hydraulic conductance, and competitive ability, while increasing root respiration. In root tips of spruce (Picea abies), O3 induces chromosomal aberrations. In leaf cells of tobacco, O3 induces genotoxicity, as shown by the Comet (alkaline single cell gel electrophoresis) Assay. Here we have used the Comet Assay in Pima cotton to show that chronic shoot exposure to ozone induced root tip genotoxicity, simultaneously with reduced gas exchange and biomass allocation to roots. The Comet Assay revealed DNA strand breaks and incomplete excision repair sites in root tips. As only the shoot is exposed to ozone this suggests that ozone impact is systemic and that translocation of either signal or cytotoxic compounds is involved in ozone phytotoxicity. This is the first application of the Comet assay to ozone damaged root tips, and the first demonstration of cytotoxic effects of ozone in roots of a crop such as cotton. While ozone injury appears to be systemic, the mechanism of shoot to root signal transduction remains to be characterized.