Salix matsudana roots exposed to 10, 50, and 100 μM Cd solutions for 24 h were carried out in order to understand the mechanisms involved in Cd tolerance and detoxification. 50 and 100 μM Cd inhibited root length significantly (P < 0.05). Cd levels in roots increased significantly with increasing Cd concentrations, and the contents of Fe, Mn, Zn, and Ca decreased significantly. A Cd-specific Leadmium Green AM dye probe showed that the meristem zone was the absorption and accumulation site of Cd in the roots. Subcellular fractionation of Cd-containing tissues indicated that about 53% of the Cd was accumulated in the cell wall of S. matsudana roots at 10 μM Cd and 65% of the Cd at 100 μM Cd, indicating that Cd binding and/or precipitation in the cell wall in roots may serve as the first barrier to reduce the cytosolic-free Cd ions. The proportion of CdE and Cdw in roots is low when compared with the other Cd chemical forms. CdHCl, Cdr, and CdHAc represent 46% (10 μM Cd), 49% (50 μM Cd), and 59% (100 μM Cd) of total Cd, and CdNaCl represents 42% (10 μM Cd), 44% (50 μM Cd), and 32% (100 μM Cd).