Evidence on the contribution of root regions with varied maturity levels in iron plaque (IP) formation and root exudation of metabolites and their consequences for uptake and bioavailability of chromium (Cr) remains unknown. Therefore, we applied combined nanoscale secondary ion mass spectrometry (NanoSIMS) and synchrotron-based techniques, micro-X-ray fluorescence (µ-XRF) and micro-X-ray absorption near-edge structure (µ-XANES) to examine the speciation and localisation of Cr and the distribution of (micro-) nutrients in rice root tip and mature region. µ-XRF mapping revealed that the distribution of Cr and (micro-) nutrients varied between root regions. Cr K-edge XANES analysis at Cr hotspots attributed the dominant speciation of Cr in outer (epidermal and sub-epidermal) cell layers of the root tips and mature root to Cr(III)-FA (fulvic acid-like anions) (58–64%) and Cr(III)-Fh (amorphous ferrihydrite) (83–87%) complexes, respectively. The co-occurrence of a high proportion of Cr(III)-FA species and strong co-location signals of 52Cr16O and 13C14N in the mature root epidermis relative to the sub-epidermis indicated an association of Cr with active root surfaces, where the dissolution of IP and release of their associated Cr are likely subject to the mediation of organic anions. The results of NanoSIMS (poor 52Cr16O and 13C14N signals), dissolution (no IP dissolution) and µ-XANES (64% in sub-epidermis >58% in the epidermis for Cr(III)-FA species) analyses of root tips may be indicative of the possible re-uptake of Cr by this region. The results of this research work highlight the significance of IP and organic anions in rice root systems on the bioavailability and dynamics of heavy metals (e.g. Cr).