AIMS: Experience in the use of whole slide imaging (WSI) for primary diagnosis in pathology is very limited. We aimed to determine the accuracy of interpretation of WSI compared with conventional light microscopy (CLM) in the diagnosis of routine gynaecological biopsies. METHODS: All gynaecological specimens (n=452) received over a 2-month period at the Department of Pathology of the Hospital Clinic of Barcelona were analysed blindly by two gynaecological pathologists, one using CLM and the other WSI. All slides were digitised in a Ventana iScan HT (Roche diagnostics) at 200×. All discrepant diagnoses were reviewed, and a final consensus diagnosis was established. The results were evaluated by weighted κ statistics for two observers. RESULTS: The level of interobserver agreement between WSI and CLM evaluations was almost perfect (κ value: 0.914; 95% CI 0.879 to 0.949) and increased during the study period: κ value 0.890; 95% CI 0.835 to 0.945 in the first period and 0.941; 95%; CI 0.899 to 0.983 in the second period. Major discrepancies (differences in clinical management or prognosis) were observed in 9 cases (2.0%). All discrepancies consisted of small lesions (8 high grade squamous intraepithelial lesions of the uterine cervix, one lymph node micrometastasis of an ovarian carcinoma) underdiagnosed or missed in the WSI or the CLM evaluation. Discrepancies with no or minor clinical relevance were identified in 3.8% of the biopsies. No discrepancy was related to the poor quality of the WSI image. CONCLUSIONS: Diagnosis of gynaecological specimens by WSI is accurate and may be introduced into routine diagnosis.