AbstractNanodiamonds are of interest as nontoxic substrates for targeted drug delivery and as highly biostable fluorescent markers for cellular tracking. Beyond optical techniques, however, options for noninvasive imaging of nanodiamonds in vivo are severely limited. Here, we demonstrate that the Overhauser effect, a proton–electron polarization transfer technique, can enable high-contrast magnetic resonance imaging (MRI) of nanodiamonds in water at room temperature and ultra-low magnetic field. The technique transfers spin polarization from paramagnetic impurities at nanodiamond surfaces to ¹H spins in the surrounding water solution, creating MRI contrast on-demand. We examine the conditions required for maximum enhancement as well as the ultimate sensitivity of the technique. The ability to perform continuous in situ hyperpolarization via the Overhauser mechanism, in combination with the excellent in vivo stability of nanodiamond, raises the possibility of performing noninvasive in vivo tracking of nanodiamond over indefinitely long periods of time.