Atypia of undetermined significance (AUS) in thyroid fine-needle aspiration (FNA) has a low to intermediate incidence of malignancy, and objective criteria could allow for improved assessment of malignancy. Consecutive thyroid FNA AUS cases with surgical excision were selected. Whole-slide images (WSIs) were evaluated for basic criteria by image analysis, including total groups and nuclear/cytoplasmic (N/C) ratio. The 44 cases encompassed 23 benign and 21 malignant entities. Seventeen (81%) of 21 malignant cases and 11 (48%) of 23 benign cases had an N/C ratio of 0.50 or less. An N/C ratio of 0.50 or less had an odds ratio for malignancy of 4.64 (P = .03). A combined category of an N/C ratio of 0.50 or less and greater than 20 cell groups had an odds ratio for malignancy of 7.0 (P = .04). Establishing defined objective WSI criteria has the potential to provide an assessment of malignancy for AUS thyroid FNA cases.