The potential of cassava (Manihot esculenta Crantz.) for simultaneous Hg and Au phytoextraction was explored by investigating Hg and Au localization in cassava roots through Micro-Proton Induced X-Ray Emission, High-Resolution Transmission Electron Microscopy (HR-TEM) and X-Ray Diffractometry (XRD). The effect of Hg and Au in the cyanogenic glucoside linamarin distribution was also investigated using Matrix Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (MALDI-FT-ICR-MS) imaging. Hg was located mainly in the root vascular bundle of plants grown in 50 or 100 μmol L-1 Hg solutions. Au was localized in the epidermis and cortex or in the epidermis and endodermis for 50 and 100 μmol L-1 Au solutions, respectively. For 50 μmol L-1 solutions of both Hg and Au, the two metals were co-localized in the epidermis. When the Hg concentrations were increased to 100 μmol L-1, Au was still localized to a considerable extent in the epidermis while Hg was located in all root parts. HR-TEM and XRD revealed that Au nanoparticles were formed in cassava roots. MALDI-FT-ICR-MS imaging showed linamarin distribution in the roots of control and plants and metal-exposed plants thus suggesting that linamarin might be involved in Hg and Au uptake and distribution.