Epicuticular waxes of three trembling aspen (Populus tremuloides Michx.) clones differing in O3 tolerance were examined over six growing seasons (1998-2003) at three bioindicator sites in the Lake States region of the USA and at FACTS II (Aspen FACE) site in Rhinelander, WI. Differences in epicuticular wax structure were determined by scanning electron microscopy and quantified by a coefficient of occlusion. Statistically significant increases in stomatal occlusion occurred for the three O3 bioindicator sites, with the higher O3 sites having the most affected stomata for all three clones as well as for all treatments including elevated CO2, elevated O3, and elevated CO2 + O3. We recorded statistically significant differences between aspen clones and between sampling period (spring, summer, fall). We found no statistically significant differences between treatments or aspen clones in stomatal frequency.