BACKGROUND: Although the molecular analysis of epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) in archived lung cancer tissues is relatively well established, the genetic testing of cytological material has not yet become a routine.METHODS: The current study used cell samples that were obtained by bronchial brushing, transthoracic needle aspiration, or biopsy imprint preparation between 1993 and 2008. Islets of malignant cells were visually located on the archived cytological slides, lysed in situ by a drop of sodium dodecyl sulfate‐containing buffer, and subjected to the standard DNA and RNA extraction. Examination of paraffin‐embedded tissue blocks (resection specimens or biopsy material) from the same patients was performed in parallel.RESULTS: A total of 75 cytological/histological lung adenocarcinoma sample pairs underwent polymerase chain reaction analysis for the EGFR mutation. Two cytological samples and 1 morphological sample failed to produce DNA. Concordance for the wild‐type and mutation status was observed in 54 of 72 and 14 of 72 informative pairs, respectively; 3 pairs and 1 pair, respectively, had mutation only in the cytological or histological material. The discrepancies could be explained by the failure to ensure a high percentage of lung cancer cells in the analyzed samples or, alternatively, by the genuine intratumoral molecular heterogeneity of some neoplasms. RNA extraction followed by reverse transcriptase‐polymerase chain reaction analysis for the EML4‐ALK translocation was performed for 44 EGFR mutation‐negative sample pairs; failures were observed for 2 cytological and 6 histological specimens. All informative pairs were concordant either for the norm (32 of 36 pairs) or for the presence of EML4‐ALK gene fusion (4 of 36 pairs).CONCLUSIONS: Archived cytological slides appear to be well suited both for EGFR and ALK analysis. Cancer (Cancer Cytopathol) 2013;121:370–376. © 2013 American Cancer Society.