Nutrient loss, specifically of nitrate and phosphate, from agricultural land often incurs harmful consequences for environmental and groundwater quality and many efforts have been attempted to reduce the discharge of agricultural nutrients to open waters and groundwater, yet no approach has shown to be robust. Biochar has been demonstrated to alter soil properties, thus soils modified with biochar having significant anion exchange capacity (AEC) may exhibit reduced nutrient loss. Our goal in this study was to determine what chemical functional groups contribute AEC to biochar and investigate what production conditions and raw material choices yield biochar with appreciable AEC. Further, we assessed stability of AEC through oxidation of biochars by exposure to singlet oxygen in an aqueous environment. We employed chemical analysis, BET-surface area, particle density and Fourier transform infra-red (FTIR) spectroscopy to characterize the various biochars. For the studied biochars, AEC values ranged from 0.602 to 27.76 cmol Kg-1 and increased with decreasing pH (P < 0.0001) and increasing pyrolysis temperature. Oxidation decreased AEC on average by 54%, however AEC of a 700 °C alfalfa meal biochar was resistant to oxidation. Higher pyrolysis temperature yielded biochar C of greater condensed aromatic character which was more resistant to loss of AEC by oxidation. Surface area and particle density did not influence AEC values. The cellulose biochar was composed almost entirely of C, H, and O but still exhibited significant AEC even at pH 8, suggesting that O containing functional groups contribute AEC. FTIR spectroscopy indicated negligible hydroxyl O, relatively small amounts of carbonyl and carboxyl O, and a prominent peak at 1590 cm-1, which we attribute to C-O+꞊C stretching in O-heterocycles. FTIR and 13C nuclear magnetic resonance (NMR) spectroscopy also revealed that biochars with different levels of condensation of aromatic C oxidize differently with biochars produced at 500 °C exhibiting development of hydroxyl and carbonyl character and biochars produced at 700 °C exhibiting peroxy ether character; hence biochar C produced at different temperatures oxidize differently. We propose possible mechanisms for oxidation. We conclude that AEC in the studied biochars is primarily due to oxonium functional groups formed during pyrolysis.