Ozone (O3) pollution affects plant growth and isoprene (ISO) emission. However, the response mechanism of isoprene emission rate (ISOrate) to elevated O3 (EO3) remains poorly understood. ISOrate was investigated in two genotypes (diploid and triploid) of Chinese white poplar (Populus tomentosa Carr.) exposed to EO3 in an open top chamber system. The triploid genotype had higher photosynthetic rate (A) and stomatal conductance (gs) than the diploid one. EO3 significantly decreased A, gs, and ISOrate of middle and lower leaves in both genotypes. In the diploid genotype, the reduction of ISOrate was caused by a systematic decrease related to ISO synthesis capacity, as indicated by decreased contents of the isoprene precursor dimethylallyl diphosphate and decreased isoprene synthase protein and activity. On the other hand, the negative effect of O3 on ISOrate of the triploid genotype did not result from inhibited ISO synthesis capacity, but from increased ISO oxidative loss within the leaf. Our findings will be useful for breeding poplar genotypes with high yield and lower ISOrate, depending on local atmospheric volatile organic compound/NOx ratio, to cope with both the rising O3 concentrations and increasing biomass demand. They can also inform the incorporation of O3 effects into process-based models of isoprene emission.