The distribution and speciation of U and Cu in contaminated vadose zone and aquifer sediments from the U.S. DOE Hanford site (300 Area) were determined using a combination of synchrotron-based micro-X-ray fluorescence (μXRF) imaging, micro-X-ray absorption near edge structure (μXANES) spectroscopy, and micro-X-ray diffraction (μXRD) techniques combined with bulk U L_III-edge X-ray absorption fine structure (XAFS) spectroscopy. Samples were collected from within the inactive North Process Pond (NPP2) at 8 ft (2.4 m, NPP2−8) depth and 12 ft (3.7 m, NPP2-12) depth in the vadose zone, and fines were isolated from turbid groundwater just below the water Table (12−14 ft, ∼4 m, NPP2-GW). μXRF imaging, μXRD, and μXANES spectroscopy revealed two major U occurrences within the vadose and groundwater zones: (1) low to moderate concentrations of U(VI) associated with fine-textured grain coatings that were consistently found to contain clinochlore (referred to here as chlorite) observed in all three samples, and (2) U(VI)−Cu(II) hotspots consisting of micrometer-sized particles associated with surface coatings on grains of muscovite and chlorite observed in samples NPP2-8′ and NPP2-GW. In the aquifer fines (NPP2-GW), these particles were identified as cuprosklodowskite (cps: Cu[(UO₂)(SiO₂OH)]₂·6H₂O) and metatorbernite (mtb: Cu(UO₂)₂(PO₄)₂·8H₂O). In contrast, the U−Cu-containing particles in the vadose zone were X-ray amorphous. Analyses of U L_III-edge XAFS spectra by linear-combination fitting indicated that U speciation consisted of (1) ∼75% uranyl sorbed to chlorite and ∼25% mtb-like X-ray amorphous U−Cu-phosphates (8 ft depth), (2) nearly 100% sorbed uranyl (12 ft depth), and (3) ∼70% uranyl sorbed to chlorite and ∼30% cps/mtb (groundwater zone). These findings suggest that dissolution of U(VI)−Cu(II)-bearing solids as well as desorption of U(VI), mainly from phyllosilicates, are important persistent sources of U(VI) to the associated uranium groundwater plume in Hanford Area 300.