Colon biopsy diagnostics may reliably be performed using virtual microscopy Objective: Virtual microscopy using whole slide images (WSIs) is a feasible alternative to optical microscopy, offering major advantages for pathology practice. The present study aims to prove reliability of this promising technique for colon biopsy diagnostics. Method: Colon biopsies (n=295) were assessed (into 7 main diagnostic groups) using both glass slides and WSIs by 4 pathologists and 2 residents. Two of the pathologists having ample experience using WSIs, scored the biopsies in a primary diagnostic setting. For each case the criterion standard diagnosis was defined based on glass slide diagnoses. Accuracy was defined as the percentage of concordance with the criterion standard. Kappa statistics were calculated as a measure of observer agreement. Results: The overall concordance rates were 89.5 % for WSIs and 91.4 % for conventional microscopy. The intraobserver (WSIs versus glass slides) agreement was good to excellent, with kappa values ranging from 0.73 to 0.87 (mean 0.78) and was higher than the interobserver agreement for glass slides (mean 0.71). Concordance with the criterion standard varied less between WSIs and glass slides in the diagnoses of pathologists with virtual experience. Conclusion: This study showed good diagnostic accuracy and reproducibility for WSIs, indicating this technology may be used for colon biopsy diagnostics.