Oral squamous cell carcinomas develop in precancerous fields consisting of genetically altered mucosal epithelial cells. These precancerous fields may appear as clinically visible lesions, in particular, oral leukoplakia, but the large majority remains clinically undetectable. The aim of this study was to assess the potential value of a noninvasive screening approach to detect precancerous fields. As a first step, we developed a suitable assay and investigated 25 leukoplakia patients and 20 noncancer control subjects. Exfoliated cells were removed by a brush from multiple small areas of the oral mucosa, including the leukoplakia. Brushed samples were investigated for allelic imbalance (AI) at chromosomes 3p, 9p, 11q, and 17p using microsatellite markers known to show frequent alterations in oral precancer. AI was absent in all (137) of the samples of the 20 control subjects, yielding a specificity of 100%. AI was detected in exfoliated cell samples of 40% (10 of 25) of the leukoplakia lesions studied. Genetic changes were also found outside the leukoplakia lesions. Most frequent was AI at 9p (9 of 10). The noninvasive assay was validated against the biopsy results of the leukoplakia lesions yielding an estimate of sensitivity of 78% (7 of 9) and a positive predictive value of 100% (7 of 7). Altogether, these results show the feasibility of a noninvasive genetic screening approach for the detection and monitoring of oral precancer. This assay could therefore contribute to the secondary prevention of oral squamous cell carcinoma. The assay also shows promise for the detection of precancerous changes that are not macroscopically visible.