To quantitatively examine the DNA content and nuclear morphometric status of oral leukoplakia (OL) and investigate its association with the degree of dysplasia in a cytologic study. Oral cytobrush biopsy was carried out to obtain exfoliative epithelial cells from lesions before scalpel biopsy at the same location in a blinded series of 70 patients with OL. Analysis of nuclear morphometry and DNA content status using image cytometry was performed with oral smears stained with the Feulgen-thionin method. Nuclear morphometric analysis revealed significant differences in DNA content amount, DNA index, nuclear area, nuclear radius, nuclear intensity, sphericity, entropy, and fractal dimension (all P<.01) between low-grade and high-grade dysplasia. DNA content analysis identified 34 patients with OL (48.6%) with DNA content abnormality. Nonhomogeneous lesion (P=.018) and high-grade dysplasia (P=.008) were significantly associated with abnormal DNA content. Importantly, the positive correlation between the degree of oral dysplasia and DNA content status was significant (P=.004, correlation coefficient=0.342). Cytology analysis of DNA content and nuclear morphometric status using image cytometry may support their use as a screening and monitoring tool for OL progression.