Objective Detection of Epstein–Barr virus (EBV) status might help in the diagnosis of EBV‐related neoplasms. The rate of successful assays for the detection of EBV‐infected cells in cytological preparations has not been fully explored. Our aims were to examine the rate of successful in situ hybridization (ISH) assays for EBV‐encoded RNA (EBER) in cytological specimens and to explore reasons for failure. Methods An electronic search selected cases with ISH‐EBER assays performed on cytological preparations during a 10‐year period. Data regarding patient age, gender and immune status, sample type and site, type of preparation, ISH‐EBER results, immunophenotyping and immunohistochemistry results, final diagnosis and correspondent histopathological samples were retrieved. Results Sixty specimens from 58 patients with diagnoses of lymphoproliferative disorder (n = 35), carcinoma (n = 24) and sarcoma (n = 1) were identified. ISH‐EBER assays were performed on 50 cell block sections and on 10 cytospin preparations, with 22 positive and 32 negative results. Six tests (four cytospins and two cell block sections) failed owing to loss of material during the assay and background staining, with an overall failure rate of 10% and 4% if cytospins were excluded. Assays were performed on 13 cytology and surgical specimens from the same site, with only one discrepant result. Conclusions Cell block sections had more successful ISH‐EBER assays when compared with cytospins. Reasons for failure were loss of material on the slide and background staining. A high concordance rate with surgical specimens emphasizes the usefulness of cytological samples for determining EBV status in patients with exhausted or no histological material available.