Abstract The humification of plant litter is a crucial step for the buildup of soil organic matter in terrestrial ecosystems. Yet, how environmental change and substrate quality affect root humification still remains poorly understood. A two-year litterbag experiment was conducted to assess the root mass remaining and accumulation of humic substances in roots of three diameter classes (0–2, 2–5 and 5–10 mm) of two common subalpine tree species (Picea asperata and Abies faxoniana) at two elevations (3037 m and 3580 m) on the eastern Tibetan Plateau. No significant differences were found between elevation treatments in the concentration of humic substances. Both root mass loss and concentrations of humic substance, humic acid and fulvic acid decreased with increasing root diameter. Both fulvic acid concentration and humification degree declined as root decomposition progressed but humic acid concentration exhibited an opposite trend. Our results reveal that the diameter-associated variations in accumulation of humic substances were substantially stronger than altitudinal and interspecific differences in decomposing root litters. These findings have important implications for carbon sequestration via root humification in the subalpine forests experiencing snow-covered winter.