Sedum alfredii is a fast-growing, high-biomass zinc (Zn) hyperaccumulator native to China. Here, the characteristics of in vivo Zn distribution in stems and leaves of the hyperaccumulating (HE) and nonhyperaccumulating ecotypes (NHE) of S. alfredii were investigated by synchrotron radiation X-ray fluorescence (SRXRF) analysis, together with a Zn probe. Preferential Zn accumulation in leaf and stem epidermis was observed in both ecotypes, but to a much greater extent for HE. Epidermal Zn increased largely in leaves and stems of HE as exposure time was prolonged, while Zn saturation occurred relatively early in HE leaf mesophyll cells and stem vascular bundles. A second peak of Zn enrichment in stem and leaf vascular systems was shown in both ecotypes. However, the proportion of Zn accumulated in stem vascular bundles relative to other tissues was much greater for HE than for NHE. Leaf and stem distribution patterns of phosphorus (P) and sulphur (S) in the HE were very like that for Zn, while the calcium (Ca) distribution pattern was the reverse of that for Zn. No such relationship was observed in NHE. Our study mainly suggested that epidermal layers serve as important storage sites for accumulated Zn in the S. alfredii HE. New Phytologist (2009) 182: 116-126doi: 10.1111/j.1469-8137.2008.02740.x.