Abstract: Objective To analyze the occurrence of EML4-ALK gene rearrangement in non-small cell lung cancer （NSCLC） detected by Ventana immunohistochemical staining （IHC）， as to explore the challenges of Ventana IHC and provide a reference for other hospital to carry out the examination. Methods In this study, 659 cases of NSCLC were retrospectively analyzed and part of samples were checked by qRT-PCR. Results The occurrence rate of EML4-ALk in adenocarcinoma was 8.78% and in squamous cell carcinoma was 4.49%. Total occurrence in NSCLC was 8.48%. All cases with IHC staining （-） and （+） were confirmed negative mutation with qRT-PCR. Five cases with IHC staining （+++） were positive confirmed by qRT-PCR. One out of 5 cases with IHC staining （++） was confirmed positive by qRT-PCR. Conclusion EML4-ALK predominantly occurred in lung adenocarcinoma. There are some challenges in diagnosis of Ventana IHC. All cases with IHC staining （++） need to be verified by qRT-PCR or other Methods .