MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYH-associated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.