Salix matsudana is a low cadmium (Cd)-accumulating willow (LCW), whereas its cultivated variety, Salix matsudana var. matsudana f. umbraculifera Rehd., is a high Cd-accumulating and tolerant willow (HCW). The physiological and molecular mechanisms underlying differential Cd accumulation and tolerance in the two Salix species are poorly understood. Here, we confirmed that the differential Cd translocation capacity from roots to the shoots leads to the differential Cd accumulation in their aboveground parts between these two willow genotypes. Cd accumulation happens preferentially in transport path and Cd is mainly located in the vacuolar, cell wall and intercellular space in HCW bark by cadmium location analysis at tissue and subcellular levels. Comparative transcriptome analysis revealed that higher expressions of several metal transporter genes (ABC transporters, K+ transporters/channels, yellow stripe-like proteins, Zinc regulated transporter/iron-regulated transporter-like proteins, etc.) are involved in root uptake and translocation capacity in HCW; meanwhile, ascorbate-glutathione metabolic pathways play essential roles in Cd detoxification and higher tolerance of the Cd-accumulator HCW. These results lay the foundation for further understanding the molecular mechanisms of Cd accumulation in woody plants, and provide new insights for molecular assisted screening woody plant varieties for phytoremediation.