This study presents a comparative assessment of the trace metal air pollutants of urban, peri-urban, and rural areas of the Brahmaputra Valley plain in the Eastern Himalayan region using biomonitoring of Pyxine cocoes. In situ collection of the thalli growing on Bombax sp. from representative locations was done, which was analyzed for Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Pb, and Zn using ICP-OES. The metals, viz. Cd, Cr, Ni, Pb, and Zn, were highly enriched, indicating anthropogenic influences. The coefficients of variation (CV) of Co, Cr, and Ni were also high, pointing at their accumulation from local sources. Influence of local sources was also observed for Cd, Fe, and Mn in peri-urban and Cd in urban samples. Metals related to automobiles were accumulated in greater volume in samples of peri-urban locations, which implies the impact of the highway that runs through these locations and other associated human activities. The samples of urban areas were found to be enriched with metals originating from both vehicular emissions and road dust. Also, accumulations of Co, Cr, Cu, Fe, Mn, and Ni in the lichen thalli were found to be around tea gardens. Inter-species correlations were found to be positively significant for most of the elements. Principal component analysis (PCA) of the metal data revealed that vehicular emission and coal burning, street dust, and crustal dust were the major sources of trace metals in the ambient air of the region.