To unravel the physiological and molecular regulation mechanisms underlying the variation in copper (Cu)accumulation, translocation and tolerance among five apple rootstocks, seedlings were exposed to either basal or excess Cu. Excess Cu suppressed plant biomass and root architecture, which was less pronounced in Malus prunifolia Borkh., indicating its relatively higher Cu tolerance. Among the five apple rootstocks, M. prunifolia exhibited the highest Cu concentration and bio-concentration factor in roots but the lowest translocation factor, indicating its greater ability to immobilize Cu and restrict translocation to the aerial parts. Higher Cu concentration in cell wall fraction but lower Cu proportion in membrane-containing and organelle-rich fractions were found in M. prunifolia. Compared with the other four apple rootstocks under excess Cu conditions, M. prunifolia had a lower increment of hydrogen peroxide in roots and leaves and malondialdehyde in roots, but higher concentrations of carbohydrates and enhanced antioxidants. Transcript levels of genes involved in Cu uptake, transport and detoxification revealed species-specific differences that are probably related to alterations in Cu tolerance. M. prunifolia had relatively higher gene transcript levels including copper transporters 2 (COPT2), COPT6 and zinc/iron-regulated transporter-related protein 2 (ZIP2), which probably took part in Cu uptake, and C-type ATP-binding cassette transporter 2 (ABCC2), copper chaperone for Cu/Zn superoxide dismutase (CCS), Cu/Zn superoxide dismutase 1 (CSD1) and metallothionein 2 (MT2) probably implicated in Cu detoxification, and relatively lower mRNA levels of yellow stripe-like transporter 3 (YSL3) and heavy metal ATPase 5 (HMA5) involved in transport of Cu to aerial parts. These results suggest that M. prunifolia is more tolerant to excess Cu than the other four apple rootstocks under the current experimental conditions, which is probably attributed to more Cu retention in roots, subcellular partitioning, well-coordinated antioxidant defense mechanisms and transcriptional expression of genes involved in Cu uptake, translocation and detoxification.