Lichens have been used as reliable indicators of air quality in a diverse range of ecosystems. Recent work attributes variation of intra-thalline morphology to air pollution, though few studies have simultaneously examined multiple environmental influences on intra-thalline morphology. Variation of intra-thalline morphology may serve as a hyper-sensitive indicator of air pollution, as well as other abiotic factors. Our study investigated correlations between NO₂ pollution, canopy stratification, and arboreal substrate pH on algal layer proportions of the pollution-tolerant lichen species Parmelia sulcata. First, we collected P. sulcata from one rural and two urban locations along a low-level NO₂ concentration gradient in the Pacific Northwest. Light microscopy revealed a significant increase in algal layer proportions in sites with an increased NO₂ concentration. Second, we collected P. sulcata at equal intervals from the base to the crown of Alnus rubra trees. The results indicate an increase in algal layer proportions with an increase in canopy height. Third, we sampled P. sulcata from two wayside boreal species: Pseudotsuga menziesii and A. rubra. The results suggest differences in algal layer thickness among substrates; algal layer proportions were generally lower on substrates with higher pH. These data suggest that multiple independent variables may impact intra-thalline morphology. Therefore, we caution against ascribing variation in algal layer ratios to pollution concentrations without accounting for potentially confounding ecological sources of variation.