Epicuticular waxes of three trembling aspen (Populus tremuloides Michx.) clones differing in O3 tolerance were examined over six growing seasons (1998–2003) at three localities (Rhinelander, WI − clean and control site; Kalamazoo, MI − moderate pollution loading and; Kenosha, WI − high pollution loading) in the Lake States regions of the USA and at the Aspen FACE site in Rhinelander, WI. Differences in epicuticular wax structure were determined by scanning electron microscopy and quantified by the coefficient of occlusion. Statistically significant increases in stomatal occlusion occurred for the three O3 bio-indicator sites, as we predicted, with the higher O3 sites having the most affected stomata for all three clones, and also for all treatments including elevated CO2, elevated O3, and elevated CO2 + O3. The results suggest that O3 pollution of the Kenosha and Kalamazoo sites show significant negative impact on epicuticular waxes of aspen, and these impacts are the most severe on the most O3 sensitive clones. We recorded statistically significant differences between aspen clones in the spring, summer and autumn sampling periods, and in the Rhinelander, Kalamazoo and Kenosha localities. However, we found no statistically significant differences in stomatal occlusion between treatments at Aspen FACE. Nutrition ratio (S/N) was disturbed in all observed tree species compared to the limit range. In all cases, our results showed that increased sulphur exceeded requirements for these plants protein synthesis. The foliage surface of three aspen clones contained Al, Si, Ca, Fe, Mg, K, Cl, Mn, Na, Ni, Ti in all the studied localities. Particles containing Th and Y were found in the Rhinelander locality, and Ba containing-particles were recorded in Kenosha.