Accumulation of arsenic (As) within plant tissues represents a human health risk, but there remains much to learn regarding the speciation of As within plants. We developed synchrotron-based fluorescence-X-ray absorption near-edge spectroscopy (fluorescence-XANES) imaging in hydrated and fresh plant tissues to provide laterally resolved data on the in situ speciation of As in roots of wheat (Triticum aestivum) and rice (Oryza sativa) exposed to 2 μM As(V) or As(III). When exposed to As(V), the As was rapidly reduced to As(III) within the root, with As(V) calculated to be present only in the rhizodermis. However, no uncomplexed As(III) was detected in any root tissues, because of the efficient formation of the As(III)-thiol complex - this As species was calculated to account for all of the As in the cortex and stele. The observation that uncomplexed As(III) was below the detection limit in all root tissues explains why the transport of As to the shoots is low, given that uncomplexed As(III) is the major As species transported within the xylem and phloem. Using fluorescence-XANES imaging, we have provided in situ data showing the accumulation and transformation of As within hydrated and fresh root tissues.