The speciation and spatial distribution of selenium (Se) in hydrated plant tissues is not well understood. Using synchrotron-based X-ray absorption spectroscopy and X-ray fluorescence microscopy [two-dimensional scanning (and associated mathematical model) and computed tomography], the speciation and distribution of toxic Se was examined within hydrated roots of cowpea (Vigna unguiculata (L.) Walp.) exposed to either 20 µM selenite or selenate. Based upon bulk solution concentrations, selenate was nine-fold more toxic to the roots than selenite, most likely due to increased accumulation of organoselenium (e.g. selenomethionine) in selenate-treated roots. Specifically, uptake of selenate (probably by sulfate transporters) occurred at a much higher rate than for selenite (apparently by both passive diffusion and phosphate transporters), with bulk root tissue Se concentrations ca. 18-fold higher in the selenate treatment. Although the proportion of Se converted to organic forms was higher for selenite (100 %) than for selenate (26 %), the absolute concentration of organoselenium was actually ca. five-fold higher for selenate-treated roots. In addition, the longitudinal and radial distribution of Se in roots differed markedly - the highest tissue concentrations were in the endodermis and cortex ca. ≥ 4 mm behind the apex when exposed to selenate, but in the meristem (ca. 1 mm from the apex) when exposed to selenite. The examination of the distribution and speciation of Se in hydrated roots provide valuable data in understanding the Se uptake, transport, and toxicity.