The bark of quaking aspen (Populus tremuloides), a clonal hardwood that is dominant in many Rocky Mountain forests, varies from white to orange to green among both ramets and populations. The proximate causes of this color variation remain controversial. We collected 72 samples of aspen bark from 11 locations in Boulder County, Colorado, and used microscopy, dissection, and thin layer chromatography to assess the structural and biochemical basis for the color gradient. Our study shows that the cork cells closest to the cork cambium of the aspen were consistently orange and conglutinated. Outward from this layer, the cork cells transitioned from orange to white; they aged and simultaneously lost cohesion. Green bark was visible due to thinnest cork layers, which revealed subcortical chlorenchyma tissue, the only bark tissue in which photosynthetic pigments were detected. Tests for photosynthetic pigmentation in the cork were negative. Comparison to standards indicated that b-carotene is not the pigment responsible for the orange hue of the cambium. We conclude that the powdery substance found on the tree surface is composed of bark cells, and their color variation seen on the aspen bark is attributed to the cell's pigment content and thickness, although the molecule responsible for cork cell coloration remains unidentified.