Oral Epstein–Barr virus (EBV) infection is associated with hairy leukoplakia and possibly other oral diseases. Many studies of oral EBV infection utilize surgical specimens. This study tested a non-invasive brush biopsy technique as an alternative to surgical biopsy to study oral EBV infection and disease. Paired, same-site, samples of tongue epithelium were obtained from research subjects, first by brush and then by surgical biopsy. Brush cells and surgical specimens were fixed and prepared for histologic sectioning and/or processed for nucleic acid extraction. Brush cell pellet sections proved equivalent to surgical specimen tissue sections for hairy leukoplakia diagnosis by routine histologic staining and EBV immunohistochemistry or in situ hybridization. Amplification of EBV sequences demonstrated superiority of the brush cells over surgical specimens for both sensitivity (90% vs. 73%) and negative predictive value (93% vs. 82%). This non-invasive brush biopsy technique should facilitate larger, prospective studies of oral EBV infection and pathogenesis.