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Background: Whole slide images (WSIs) involve digitally capturing glass slides for microscopic computer-based viewing and these are amenable to quantitative image analysis. Bile duct (BD) brushing can show morphologic features that are categorized as indeterminate for malignancy. The study aims to evaluate quantitative morphologic features of atypical categories of BD brushing by WSI analysis for the identification of criteria predictive of malignancy., Materials and Methods: Over a 3-year period, BD brush specimens with indeterminate diagnostic categorization (atypical to suspicious) were subjected to WSI analysis. Ten well-visualized groups with morphologic atypical features were selected per case and had the quantitative analysis performed for group area, individual nuclear area, the number of nuclei per group, N: C ratio and nuclear size differential., Results: There were 28 cases identified with 17 atypical and 11 suspicious. The average nuclear area was 63.7 µm(2) for atypical and 80.1 µm(2) for suspicious (+difference 16.4 µm(2); P = 0.002). The nuclear size differential was 69.7 µm(2) for atypical and 88.4 µm(2) for suspicious (+difference 18.8 µm(2); P = 0.009). An average nuclear area >70 µm(2) had a 3.2 risk ratio for suspicious categorization., Conclusion: The quantitative criteria findings as measured by image analysis on WSI showed that cases categorized as suspicious had more nuclear size pleomorphism (+18.8 µm(2)) and larger nuclei (+16.4 µm(2)) than those categorized as atypical. WSI with morphologic image analysis can demonstrate quantitative statistically significant differences between atypical and suspicious BD brushings and provide objective criteria that support the diagnosis of carcinoma.